Expression of Hexose Kinase in Recombinant Host Cells

ABSTRACT

The invention relates to a recombinant host cell having (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. Additionally, the invention relates to methods of making and using such recombinant host cells including, for example, methods of increasing glucose consumption, methods of improving redox balance, and/or methods of increasing the production of a product of a pyruvate-utilizing pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/980,607, filed Dec. 29, 2010, which claims the benefit of priority of U.S. Provisional Application No. 61/290,639, filed Dec. 29, 2009. The entirety of each is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name: 20140123_CL4894USCNT_SQL_ascii.txt; Size: 422,596 bytes; and Date of Creation: Jan. 23, 2014, filed herewith, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of industrial microbiology and alcohol production. More specifically, the invention relates to a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in said recombinant host cell; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. Additionally, the invention relates to methods of making and using such a recombinant host cell including, for example, methods of increasing glucose consumption, methods of enhancing redox balance, and methods of increasing the production of a product of a pyruvate-utilizing pathway.

BACKGROUND OF THE INVENTION

Global demand for liquid transportation fuel is projected to strain the ability to meet certain environmentally driven goals, for example, the conservation of oil reserves and limitation of greenhouse gas emissions. Such demand has driven the development of technology which allows utilization of renewable resources to mitigate the depletion of oil reserves and to minimize greenhouse gas emissions.

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

Methods for the chemical synthesis of isobutanol, an isomer of butanol, are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly. The production of isobutanol from plant-derived raw materials would minimize greenhouse gas emissions and would represent an advance in the art.

2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). 2,3-butanediol can be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol can be used as plasticizers (Voloch et al., “Fermentation Derived 2,3-Butanediol,” in Comprehensive Biotechnology, Pergamon Press Ltd., England, Vol. 2, Section 3:933-947 (1986)).

Microorganisms can be engineered for the expression of biosynthetic pathways that utilize pyruvate to produce, for example, 2,3-butanediol, 2-butanone, 2-butanol and isobutanol. U.S. Patent Application Publication No. US 2007/0092957 A1 discloses the engineering of recombinant microorganisms for production of isobutanol. U.S. Patent Application Publication Nos. US 2007/0259410 A1 and US 2007/0292927 A1 disclose the engineering of recombinant microorganisms for production of 2-butanone or 2-butanol. Multiple pathways are disclosed for biosynthesis of isobutanol and 2-butanol, all of which initiate with cellular pyruvate. Butanediol is an intermediate in the 2-butanol pathway disclosed in U.S. Patent Application Publication No. US 2007/0292927 A1.

Engineering recombinant host cells for increased availability of pyruvate and/or for reduced glucose repression allows for increased formation of the products of pyruvate-utilizing biosynthetic pathways. For example, reducing glucose repression has been used to improve the respiratory capacity of yeast and to increase biomass production. Also, International Publication No. WO 1998/26079 A1 discloses overexpression of the Hap1 transcription factor to reduce glucose repression results in increased respiratory capacity and increased biomass production. European Patent No. 1728854 discloses a process for biomass production using yeast overexpressing the Hap1 transcription factor grown in aerobic conditions.

Functional deletion of the hexokinase 2 gene has been used to reduce glucose repression and to increase the availability of pyruvate for utilization in biosynthetic pathways. For example, International Publication No. WO 2000/061722 A1 discloses the production of yeast biomass by aerobically growing yeast having one or more functionally deleted hexokinase 2 genes or analogs. In addition, Rossell et al. (Yeast Research 8:155-164 (2008)) found that Saccharomyces cerevisiae with a deletion of the hexokinase 2 gene showed 75% reduction in fermentative capacity, defined as the specific rate of carbon dioxide production under sugar-excess and anaerobic conditions. After starvation, the fermentation capacity was similar to that of a strain without the hexokinase 2 gene deletion. Diderich et al. (Applied and Environmental Microbiology 67:1587-1593 (2001)) found that S. cerevisiae with a deletion of the hexokinase 2 gene had lower pyruvate decarboxylase activity.

Functional deletion of the pyruvate decarboxylase gene has also been used to increase the availability of pyruvate for utilization in biosynthetic pathways. For example, U.S. Application Publication No. US 2007/0031950 A1 discloses a yeast strain with a disruption of one or more pyruvate decarboxylase genes and expression of a D-lactate dehydrogenase gene, which is used for production of D-lactic acid. U.S. Application Publication No. US 2005/0059136 A1 discloses glucose tolerant two carbon source independent (GCSI) yeast strains with no pyruvate decarboxylase activity, which may have an exogenous lactate dehydrogenase gene, Nevoigt and Stahl (Yeast 12:1331-1337 (1996)) describe the impact of reduced pyruvate decarboxylase and increased NAD-dependent glycerol-3-phosphate dehydrogenase in Saccharomyces cerevisiae on glycerol yield. U.S. patent application Ser. No. 12/477,942 discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity.

There remains a need to improve redox balance, glucose consumption and/or product formation of a pyruvate-utilizing biosynthetic pathway in recombinant host cells comprising a functional deletion of genes encoding dual-role hexokinases such as the hexokinase 2 gene.

BRIEF SUMMARY OF THE INVENTION

Provided herein are recombinant yeast cells comprising: (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in the host cell wherein the activity of the polypeptide of (a) is reduced or substantially eliminated; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity. In embodiments, the recombinant yeast cells have increased glucose consumption rates as compared to yeast cells with (a) but not (b). In embodiments, the modification of (a) is a deletion. In embodiments, the recombinant yeast cells have altered glucose repression as compared to yeast cells with (a) but not (b). In embodiments, the recombinant yeast cell further comprises (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In embodiments, pyruvate decarboxylase activity is reduced or substantially eliminated. In embodiments, the polypeptide of (a) is HXK2, and the recombinant yeast cell is S. cerevisiae. In embodiments, the polypeptide of (a) is RAG5, and the recombinant host cell is K. lactis; or the polypeptide of (a) is HPGLK1, and the recombinant host cell is H. polymorpha; or the polypeptide of (a) is HXK2, and the recombinant host cell is S. pombe. In another aspect of the invention, a polynucleotide or polypeptide of (b) corresponds to Enzyme Commission Number EC 2.7.1.1 and/or corresponds to Enzyme Commission EC 2.7.1.2. In embodiments, the polynucleotide of (b) contains a promoter such that the polypeptide of (b) is conditionally expressed. In embodiments, the conditional promoter comprises a sequence derived from the OLE1 promoter region. In embodiments, the polynucleotide of (b) contains a promoter such that the polypeptide of (b) is constitutively expressed. In embodiments, the heterologous polynucleotide of (b) comprises the polypeptide of (a) with a deletion of a protein interaction domain that prevents function as a transcriptional regulator. In embodiments, the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 2, 115, 117, 119, 4, 6, 8, 121, or 123. In embodiments, the heterologous polynucleotide of (b) comprises i) a promoter region derived from the S. cerevisiae ADH1 promoter region or ii) a promoter region having at least about 35% identity to SEQ ID NO: 131. In embodiments, the yeast cell is S. cerevisiae and the heterologous polynucleotide of (b) encodes a polypeptide of SEQ ID NO: 4, 6, 8, 121, or 123 or the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 4, 6, 8, 121, or 123. In embodiments, the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 130. In embodiments, the heterologous polynucleotide of (b) comprises a conditional promoter and encodes a polypeptide having at least 85% identity to SEQ ID NO: 4 or SEQ ID NO: 2.

One aspect of the invention relates to a recombinant host cell disclosed herein that expresses a pyruvate-utilizing biosynthetic pathway. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway comprises a heterologous polynucleotide. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway forms a product selected from 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malate, isoamyl alcohol, and isoprenoids. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (iv) 2-ketoisovalerate to isobutyraldehyde; and/or (v) isobutyraldehyde to isobutanol. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway is a 2-butanone biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; and/or (iv) 2,3-butanediol to 2-butanone. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway is a 2-butanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; and/or (v) 2-butanone to 2-butanol. In another aspect of the invention, such a pyruvate-utilizing biosynthetic pathway is a 1-butanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) acetyl-CoA to acetoacetyl-CoA; (ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA; (iii) 3-hydroxybutyryl-CoA to crotonyl-CoA; (iv) crotonyl-CoA to butyryl-CoA; (v) butyryl-CoA to butyraldehyde; and/or (vi) butyraldehyde to 1-butanol.

One aspect of the invention relates to methods for the production of a product selected from 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malic acid, isoamyl alcohol, and isoprenoids comprising (a) growing a recombinant host cell disclosed herein under conditions wherein a product is produced; and (b) optionally recovering the product. In another aspect of the invention, such methods comprise an isobutanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (iv) 2-ketoisovalerate to isobutyraldehyde; and/or (v) isobutyraldehyde to isobutanol. In another aspect of the invention, such methods comprise a 2-butanone biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; and/or (iv) 2,3-butanediol to 2-butanone. In another aspect of the invention, such methods comprise a 2-butanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; and/or (v) 2-butanone to 2-butanol. In another aspect of the invention, such methods comprise a 1-butanol biosynthetic pathway comprising a polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of (i) acetyl-CoA to acetoacetyl-CoA; (ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA; (iii) 3-hydroxybutyryl-CoA to crotonyl-CoA; (iv) crotonyl-CoA to butyryl-CoA; (v) butyryl-CoA to butyraldehyde; and/or (vi) butyraldehyde to 1-butanol.

One aspect of the invention relates to methods of producing a recombinant host cell comprising (i) providing a recombinant host cell comprising a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; and (ii) transforming a recombinant host cell of (i) with a heterologous polynucleotide encoding a polypeptide having hexose kinase activity. In another aspect of the invention, such methods further comprise (iii) introducing a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity.

One aspect of the invention relates to methods of increasing glucose consumption of a recombinant host cell comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form. In another aspect of the invention, the glucose consumption of such a recombinant host cell is greater than the glucose consumption of a recombinant host cell comprising (a) but not (b).

One aspect of the invention relates to methods of increasing glucose consumption of a recombinant host cell comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form. In another aspect of the invention, the glucose consumption of such a recombinant host cell is greater than the glucose consumption of a recombinant host cell comprising (a) and (c) but not (b).

One aspect of the invention relates to methods of improving the redox balance of a recombinant host cell comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form. In another aspect of the invention, the redox balance of such a recombinant host cell is improved compared to the redox balance of a recombinant host cell comprising (a) but not (b).

One aspect of the invention relates to methods of improving the redox balance of a recombinant host cell comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form. In another aspect of the invention, the redox balance of such a recombinant host cell is improved compared to the redox balance of a recombinant host cell comprising (a) and (c) but not (b).

One aspect of the invention relates to methods of increasing the formation of a product of a pyruvate-utilizing biosynthetic pathway comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the product of the pyruvate-utilizing pathway is formed. In another aspect of the invention, the amount of product formed by such a recombinant host cell is greater than the amount of product formed by a recombinant host cell comprising (a) but not (b). In another aspect of the invention, the product is isobutanol, 2-butanol, or 1-butanol.

One aspect of the invention relates to methods of increasing the formation of a product of a pyruvate-utilizing biosynthetic pathway comprising (i) providing a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity; and (ii) growing the recombinant host cell of (i) under conditions wherein the product of the pyruvate-utilizing pathway is formed. In another aspect of the invention, the amount of product formed by such a recombinant host cell is greater than the amount of product formed by a recombinant host cell comprising (a) and (c) but not (b). In another aspect of the invention, the product is isobutanol, 2-butanol, or 1-butanol.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The sequences in the accompanying sequence listing, filed electronically herewith and incorporated herein by reference, conform with 37 C.F.R. 1.821-1.825 (“Requirements for patent applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NOs: 1-2 and 114-119 are example dual-role hexokinases in Saccharomyces cerevisiae, described in Table 3.

SEQ ID NOs: 3-8 and 120-123 are example hexose kinase coding regions and proteins, described in Table 4.

SEQ ID NOs: 9-28 are pyruvate decarboxylase sequences described in Table 5.

SEQ ID NO: 30 is a sequence derived from the CUP1 promoter region.

SEQ ID NOs: 31 and 32 are B. subtilis acetolactate synthase coding region and protein sequences.

SEQ ID NOs: 33-36 are sequences derived from the CYC1 terminator region, ILV5 promoter region, ILV5 terminator region, and FBA1 promoter region, respectively.

SEQ ID NOs: 37 and 38 are the Pf5.IlvC-Z4B8 coding region and protein sequences.

SEQ ID NOs: 39 and 40 are the ILV5 coding region and protein sequences.

SEQ ID NOs: 41 and 42 are the Pf5.IlvC-JEA1 coding region and protein sequences.

SEQ ID NO: 44 and 47 is the L. lactis kivD coding region sequence codon optimized for S. cerevisiae and the encoded protein.

SEQ ID NO: 45 and 46 is the horse liver ADH coding region sequence codon optimized for S. cerevisiae and the encoded protein.

SEQ ID NO: 49, 53, and 54 are sequences derived from the TDH3 promoter region, GPM1 promoter region, and ADH1 terminator region, respectively.

SEQ ID NOs: 55 and 56 are the sadB coding region and protein sequences, respectively.

SEQ ID NOs: 60 and 61 are FBA terminator region derived and CYC1 terminator region derived sequences.

SEQ ID NOs: 62 and 63 are the ilvD coding region and protein sequences, respectively.

SEQ ID NOs: 124 and 125 are the nucleic acid and amino acid sequences of KIGlk1 from K. lactis.

SEQ. ID NOs: 126 and 127 are the nucleic acid and amino acid sequences of HPHXK1 from Hansenula polymorpha.

SEQ ID NO: 131 is an ADH1 promoter region derived sequence.

SEQ ID NO: 140 and 141 are SNO1 and SNZ1 promoter region derived sequences.

SEQ ID NOs: 50-51, 57-58, 66-75, 77-80, 82-100, 104-105, 107-109, 112-113, 129, and 133-138 are primers used in the Examples.

The following correspond to synthetic constructs:

SEQ ID NO: 29 is the sequence of pLH475-Z4B8 plasmid.

SEQ ID NO: 43 is the sequence of the pLH468 plasmid.

SEQ ID NO: 48 is the sequence of vector pNY8.

SEQ ID NO: 52 is the sequence of vector pRS425::GPM-sadB.

SEQ ID NO: 59 is the sequence of pRS423 FBA ilvD(Strep).

SEQ ID NO: 64 is the GPM-sadB-ADHt segment sequence.

SEQ ID NO: 65 is the pUC19-URA3r sequence.

SEQ ID NO: 76 is the pdc1::PPDC1-ilvD-FBA1t-URA3r integration cassette sequence.

SEQ ID NO: 81 is the sequence of his3::URA3r2 cassette.

SEQ ID NO: 102 is the sequence of pUC19::loxP-URA3-loxP.

SEQ ID NO: 103 is the sequence of pLA25.

SEQ ID NO: 106 is the sequence of pLA31.

SEQ ID NO: 110 is the sequence of pRS423::PGAL1-cre.

SEQ ID NO: 111 is the sequence of pLA32.

SEQ ID NO: 128 is the pLH475-JEA1 plasmid.

SEQ ID NO: 130 is the HXK2(DLys6-Met15) sequence.

SEQ ID NO: 132 is a codon-optimized sequence encoding HXK2 with an internal deletion of the Lys6-Met15 region with ADH1 terminator region derived sequence.

SEQ ID NO: 139 is the sequence of pUC19::loxP-URA3-loxP-HXK2(Lys6-Met15)-ADH1t.

SEQ ID NO: 142 is the sequence of pLH467.

SEQ ID NO: 143 is the sequence of pLH435.

SEQ ID NO: 144 is the sequence of pLH441

BRIEF DESCRIPTION OF

The various embodiments of the invention can be more fully understood from the following detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.

FIG. 1 depicts the growth (FIG. 1A) and isobutanol production (FIG. 1B) of a hexokinase 2 deletion yeast strain (NYLA84 [pLH468/pLH475-Z4B8]) as compared to a yeast strain without hexokinase 2 deletion (NYLA74 [pLH468/pLH475-Z4B8]), as described in Example 3.

FIG. 2 depicts a comparison of growth and isobutanol production for a strain with a hexokinase 2 deletion (NYLA84[pLH468/pLH475-Z4B8]; FIG. 2B) and a strain without hexokinase 2 deletion (NYLA74 [pLH468/pLH475-Z4B8]; FIG. 2A).

FIG. 3 depicts the specific productivity of a strain with a hexokinase 2 deletion (NYLA84 [pLH468/pLH475-Z4B8] and a strain without hexokinase 2 deletion (NYLA74 [pLH468/pLH475-Z4B8]) measured in grams of isobutanol produced per gram of cells over time.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

DETAILED DESCRIPTION OF THE INVENTION

This invention addresses the need for improved processes for the conversion of plant-derived raw materials to a product stream useful as a liquid transportation fuel. Such processes would satisfy both fuel demands and environmental concerns. Applicants have provided a means to improve redox balance, glucose consumption, and/or product formation of a pyruvate-utilizing biosynthetic pathway in a recombinant host cell comprising a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity by introducing a heterologous polynucleotide encoding a polypeptide having hexose kinase activity. Such cells exhibit improved redox balance, increased glucose consumption, and/or increased product formation of a pyruvate-utilizing biosynthetic pathway compared to a recombinant host cell comprising a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity without the introduction of a heterologous polynucleotide encoding a polypeptide having hexose kinase activity. Applicants have also provided methods of making and using such a recombinant host cell including, for example, methods of improving redox balance, methods of increasing glucose consumption, and methods of increasing the production of a product of a pyruvate-utilizing biosynthetic pathway.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference, unless only specific sections of patents or patent publications are indicated to be incorporated by reference.

Although methods and materials similar or equivalent to those disclosed herein can be used in practice or testing of the present invention, suitable methods and materials are disclosed below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

In order to further define this invention, the following terms, abbreviations and definitions are provided.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as disclosed in the application.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “butanol” as used herein, refers to 2-butanol, 1-butanol, isobutanol, or mixtures thereof.

The term “pyruvate-utilizing biosynthetic pathway” refers to an enzyme pathway to produce a biosynthetic product from pyruvate.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to an enzyme pathway to produce 2-butanone from pyruvate.

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term “1-butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol from pyruvate.

The terms “hxk2 mutant,” “HXK2 knockout,” or “HXK2-KO” as used herein refer to a S. cerevisiae host cell that has a genetic modification to inactivate or reduce expression of a gene encoding hexokinase 2 so that the cell substantially or completely lacks hexokinase 2 enzyme activity.

The terms “Mc mutant,” “PDC knockout,” or “PDC-KO” as used herein refer to a cell that has a genetic modification to inactivate or reduce expression of a gene encoding pyruvate decarboxylase (Pdc) so that the cell substantially or completely lacks pyruvate decarboxylase enzyme activity. If the cell has more than one expressed (active) PDC gene, then each of the active PDC genes may be inactivated or have minimal expression.

The term “carbon substrate” refers to a carbon source capable of being metabolized by the recombinant host cells disclosed herein. Non-limiting examples of carbon substrates are provided herein and include, but are not limited to, monosaccharides including, but not limited to, glucose, fructose, xylose, and arabinose; oligosaccharides including, but not limited to, sucrose and maltose; polysaccharides: and non-carbohydrate carbon sources including, but not limited to, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, or mixtures thereof.

The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5′ and 3′ sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. “Polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

A polynucleotide sequence may be referred to as “isolated,” in which it has been removed from its native environment. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. An isolated polynucleotide fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.

As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5c non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

As used herein, “hexose kinase activity” refers to the activity of any polypeptide having a biological function of a hexose kinase, including the examples provided herein. Such polypeptides include glucokinases and hexokinases. Such polypeptides also include a polypeptide that catalyzes the conversion of hexose to hexose-6-phosphate, the conversion of D-glucose to D-glucose 6-phosphate, O-fructose to O-fructose 6-phosphate, and D-mannose to D-mannose 6-phosphate. Such polypeptides also include a polypeptide that corresponds to Enzyme Commission Number EC 2.7.1.1 or to Enzyme Commission Number EC 2.7.1.2. Such polypeptides can be determined by methods well known in the art and disclosed herein.

As used herein, “hexokinase 2 activity” refers to the activity of any polypeptide having a biological function of a Saccharomyces cerevisiae hexokinase 2 enzyme, including the examples provided herein. Such polypeptides include a polypeptide that catalyzes the conversion of hexose to hexose-6-phosphate, the conversion of D-glucose to D-glucose 6-phosphate, D-fructose to D-fructose 6-phosphate, and D-mannose to D-mannose 6-phosphate. Such polypeptides also include a polypeptide that corresponds to Enzyme Commission Number EC 2.7.1.1. Such polypeptides can be determined by methods well known in the art.

As used herein, “dual-role hexokinase activity” refers to the activity of any polypeptide having a biological function of a hexose kinase enzyme and exerting a glucose repression phenotype in the cell in which it is expressed. Such polypeptides include a polypeptide that catalyzes the conversion of hexose to hexose-6-phosphate, the conversion of D-glucose to D-glucose 6-phosphate, D-fructose to D-fructose 6-phosphate, and D-mannose to D-mannose 6-phosphate. The second role that a hexose kinase may have is regulatory: A hexokinase is dual-role in a yeast host if it functions to exert glucose repression on glucose-repressible genes. This may be demonstrated by relief from glucose repression in a strain with a mutation in the gene encoding that hexokinase. The dual-role is specific to a particular host cell, thus, a hexose kinase having both hexose kinase activity and glucose repression activity in one species may not express the glucose repression function in another. Hexose kinases including dual-function hexokinases are known in the art.

As used herein, “pyruvate decarboxylase activity” refers to any polypeptide having a biological function of a pyruvate decarboxylase enzyme, including the examples provided herein. Such polypeptides include a polypeptide that catalyzes the conversion of pyruvate to acetaldehyde. Such polypeptides also include a polypeptide that corresponds to Enzyme Commission Number 4.1.1.1. Such polypeptides can be determined by methods well known in the art and disclosed herein.

As used herein, “reduced activity” refers to any measurable decrease in a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the reduced activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. A reduced activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.

As used herein, “substantially eliminated activity” refers to measurable decrease in a known biological activity of a polypeptide that results in nearly complete abolishment of the activity when compared to the same biological activity of the polypeptide prior to the change resulting in the substantially eliminated activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. A substantially eliminated activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.

As used herein, “eliminated activity” refers to the complete abolishment of a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. An eliminated activity includes a biological activity of a polypeptide that is not measurable when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity. An eliminated activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.

By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

As used herein, “native” refers to the form of a polynucleotide, gene or polypeptide as found in nature with its own regulatory sequences, if present.

As used herein, “endogenous” refers to the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism. “Endogenous polynucleotide” includes a native polynucleotide in its natural location in the genome at an organism. “Endogenous gene” includes a native gene in its natural location in the genome of an organism. “Endogenous polypeptide” includes a native polypeptide in its natural location in the organism.

As used herein, “heterologous” refers to a polynucleotide, gene or polypeptide not normally found in the host organism but that is introduced into the host organism or is otherwise modified from its native state. “Heterologous polynucleotide” includes a native coding region from the host organism, or portion thereof, that is reintroduced into or is otherwise modified from the host organism in a form that is different from the corresponding native polynucleotide as well as a coding region from a different organism, or portion thereof. “Heterologous gene” includes a native coding region, or portion thereof, that is reintroduced or otherwise modified in the source organism in a form that is different from the corresponding native gene as well as a coding region from a different organism. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. “Heterologous polypeptide” includes a native polypeptide that is in a form that is different from the corresponding native polypeptide as well as a polypeptide from another organism. A polypeptide that is altered such that the expression pattern (such as transcriptional or translational profile or cellular localization) is different from that of the native polypeptide is considered heterologous.

As used herein, the term “modification” refers to a change in a polynucleotide or polypeptide that results in reduced, substantially eliminated or eliminated activity of a polypeptide encoded by the polynucleotide, as well as a change in a polypeptide disclosed herein that results in reduced, substantially eliminated or eliminated activity of the polypeptide. Such changes can be made by methods well known in the art, including, but not limited to, deleting, mutating (e.g., spontaneous mutagenesis, random mutagenesis, mutagenesis caused by mutator genes, or transposon mutagenesis), substituting, inserting, down-regulating, altering the cellular location, altering the state of the polynucleotide or polypeptide (e.g., methylation, phosphorylation or ubiquitination), removing a cofactor, introduction of an antisense RNA/DNA, introduction of an interfering RNA/DNA, chemical modification, covalent modification, irradiation with UV or X-rays, homologous recombination, mitotic recombination, promoter replacement methods, and/or combinations thereof. Guidance in determining which nucleotides or amino acid residues can be modified can be found by comparing the sequence of the particular polynucleotide or polypeptide with that of homologous polynucleotides or polypeptides, e.g., yeast or bacterial, and maximizing the number of modifications made in regions of high homology (conserved regions) or consensus sequences. Other modifications to polynucleotides may result in increased expression, such as in the case of biosynthetic pathways for the production of a product.

As used herein, the term “variant” refers to a polypeptide differing from a specifically recited polypeptide of the invention by amino acid insertions, deletions, mutations, and substitutions, created using, e.g., recombinant DNA techniques, such as mutagenesis. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, e.g., yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.

Alternatively, recombinant polynucleotide variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.

Amino acid “substitutions” may be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they may be the result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, “non-conservative” amino acid substitutions may be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. “Insertions” or “deletions” may be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

The term “overexpression,” as used herein, refers to expression that is higher than endogenous expression of the same or related gene. A heterologous gene is overexpressed if its expression is higher than that of a comparable endogenous gene.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

As used herein the terms “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TCC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L) TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S) TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met ACG Thr (T) AAG Lys (K) AGG Arg (R) (M) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can be adapted in a number of ways, See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. Table 2 has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons,

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Frequency per Amino Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 120510 18.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC 35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 Ile AUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 144243 22.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU 153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 Ser AGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8 Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 83207 12.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC 82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 Tyr UAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3 Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA 273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2 Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 31095 4.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 19562 3.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU 156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 Stop UAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the Vector NTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the “backtranslation” function at http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visited Apr. 15, 2008) and the “backtranseq” function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as “synthetic gene designer” (http//phenotype.biosci.umbc.edu/codon/sgd/index.php).

A polynucleotide or nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference) The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with aspect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as provided herein, as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those disclosed in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (disclosed by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, such as from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencer (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al, Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).

The genetic manipulations of a recombinant host cell disclosed herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable for genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In embodiments, a recombinant host cell disclosed herein can be any yeast or fungi host useful for genetic modification and recombinant gene expression. In other embodiments, a recombinant host cell can be a member of the genera Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Issatchenkia, Brettanomyces, Torulaspora, Hanseniaspora, Kluyveromyces, and some species of Candida. In another embodiment, a recombinant host cell can be S. cerevisiae.

Modification of Dual-Role Hexokinase

Recombinant yeast cells disclosed herein can comprise a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in said host cell and/or a modification in a polypeptide having dual-role hexokinase activity in said host cell. In embodiments, a recombinant host cell disclosed herein can have a modification or disruption of one or more polynucleotides, genes or polypeptides encoding dual-role hexokinases. In embodiments, a recombinant host cell comprises a deletion, mutation, and/or substitution in one or more endogenous polynucleotides or genes encoding a polypeptide having dual-role hexokinase activity, or in one or more endogenous polypeptides having dual-role hexokinase activity. Such modifications, disruptions, deletions, mutations, and/or substitutions can result in dual-role hexokinase activity that is reduced or substantially eliminated, resulting, for example, in a dual-role hexokinase knockout phenotype.

In embodiments, a polypeptide having dual-role hexokinase activity can catalyze the conversion of hexose to hexose-6-phosphate, and/or can catalyze the conversion of D-glucose to D-glucose 6-phosphate, D-fructose to D-fructose 6-phosphate, and/or D-mannose to D-mannose 6-phosphate. In other embodiments, a polynucleotide, gene or polypeptide having dual-role hexokinase activity can correspond to Enzyme Commission Number EC 2.7.1.1.

In embodiments, a recombinant host cell can be S. cerevisiae and a polynucleotide, gene or polypeptide having dual-role hexokinase activity can be hexokinase 2 (HXK2). In embodiments, a recombinant host cell can be K. lactis and a polynucleotide, gene or polypeptide having dual-role hexokinase activity can be RAG5. In other embodiments, a recombinant host cell can be H. polymorpha and a polynucleotide, gene or polypeptide having dual-role hexokinase activity can be HPGLK1. In other embodiments, a recombinant host cell can be S. pombe and a polynucleotide, gene or polypeptide having dual-role hexokinase activity can be HXK2. Hexokinase 2 knockout strains are known in the art (Vojtek and Fraenkel, Eur. J. Biochem. 190: 371-375, 1990; Lobo and Maitra, Genetics 86: 727-744, 1977; Winzeler, et al. Science 285: 901-906, 1999; and American Type Culture Collection #4004620, #4014820, #4024620, and #4034620).

Other examples of dual-role hexokinase polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, dual-role hexokinase polynucleotides, genes and/or polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of the sequences disclosed herein, wherein such a polynucleotide or gene encodes, or such a polypeptide has, dual-role hexokinase activity. Still other examples of dual-role hexokinase polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, an active variant, fragment or derivative of any one of the sequences disclosed herein, wherein such a polynucleotide or gene encodes, or such a polypeptide has, dual-role hexokinase activity.

In embodiments, the sequences of other dual-role hexokinase polynucleotides, genes and/or polypeptides can be identified in the literature and candidates can be identified in bioinformatics databases well known to the skilled person using sequences disclosed herein and available in the art. For example, such sequences can be identified through BLAST searching of publicly available databases with known hexose kinase encoding polynucleotide or polypeptide sequences. In such a method, identities can be based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, the dual-role hexokinase polynucleotide or polypeptide sequences disclosed herein or known the art can be used to identify other candidate hexose kinase homologs in nature. For example, each of the hexose kinase encoding nucleic acid fragments disclosed herein can be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to (1) methods of nucleic acid hybridization; (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. at al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and (3) methods of library construction and screening by complementation.

Whether or not a particular hexose kinase is a dual-role hexokinase is specific to the host cell in which the hexose kinase is expressed. For example, while Hansenula polymorpha HPGLK1 is a dual-role hexokinase in the native organism, it is not associated with glucose repression in S. cerevisiae. Additional examples of hexose kinases that are dual-role in S. cerevisiae are given in Table 3. The dual-role nature of certain hexose kinases is known in the art, and, whether or not a hexose kinase is a dual-role hexokinase in a particular host cell can be readily determined from the art and/or using methods known to those of skill in the art. For example, one of the roles of any hexose kinase is enzymatic activity to phosphorylate hexoses, as per E.C. definition 2.7.1.1 or 2.7.1.2, and such activity can be confirmed by assays known in the art. The second role that a dual-role hexokinase will have is regulatory: that is, it is exerts glucose repression on glucose-repressible genes. This is demonstrated by relief from glucose repression in a strain with a mutation in the gene encoding that hexose kinase. Glucose repression relief in the mutant strain can be demonstrated by methods known in the art, including, but not limited, to:

1. measuring expression of the enzymatic activity of an enzyme(s) known to be glucose-repressed in that host (e.g. in S. cerevisiae, invertase; maltase; galactokinase) when the cells are grown in glucose-containing medium (if the genetic system involves induction as well as repression, the cognate non-glucose carbon source must be added too, e.g. galactose, maltose);

2. measuring transcription of a gene(s) known to be glucose-repressed in that host when the cells are grown in glucose-containing medium (if the genetic system involves induction as well as repression, the cognate non-glucose carbon source must be added too, e.g. galactose, maltose). Transcription can be measured by Northern blot, RT-PCR, run-on transcription, etc. Transcription can be measured by expression of a reporter gene (e.g. GFP, lacZ, gusB) placed under control of a promoter from a glucose-repressible gene;

3. measuring the ability of the mutant strain to co-consume glucose and a carbon source whose consumption is normally repressed by glucose (e.g. in S. cerevisiae: sucrose, maltose, galactose);

4. testing the ability of the mutant strain to grow on a carbon source whose consumption is normally repressed by glucose, when the growth medium also contains a gratuitous glucose repressor (e.g. 2-deoxyglucose, 5-thioglucose).

All of the tests mentioned above could be done with the non-mutant strain as well, for reference.

In embodiments, dual-role hexokinase polynucleotides, genes and/or polypeptides related to a recombinant host cell disclosed herein can be modified or disrupted. Many methods for genetic modification and disruption of target genes to reduce or eliminate expression are known to one of ordinary skill in the art and can be used to create a recombinant host cell disclosed herein. Modifications that can be used include, but are not limited to, deletion of the entire gene or a portion of the gene encoding a dual-role hexokinase protein, inserting a DNA fragment into the encoding gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less active protein is expressed. In other embodiments, expression of a target gene can be blocked by expression of an antisense RNA or an interfering RNA, and constructs can be introduced that result in cosuppression. In other embodiments, the synthesis or stability of the transcript can be lessened by mutation. In embodiments, the efficiency by which a protein is translated from mRNA can be modulated by mutation. All of these methods can be readily practiced by one skilled in the art making use of the known or identified sequences encoding target proteins.

In other embodiments, DNA sequences surrounding a target dual-role hexokinase coding sequence are also useful in some modification procedures and are available, for example, for yeast such as Saccharomyces cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. An additional non-limiting example of yeast genomic sequences is that of Candida albicans, which is included in GPID #10771, #10701 and #16373. Other yeast genomic sequences can be readily found by one of skill in the art in publicly available databases.

In other embodiments, DNA sequences surrounding a target dual-role hexokinase coding sequence can be useful for modification methods using homologous recombination. In a non-limiting example of this method, dual-role hexokinase gene flanking sequences can be placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the dual-role hexokinase gene. In another non-limiting example, partial dual-role hexokinase gene sequences and dual-role hexokinase gene flanking sequences bounding a selectable marker gene can be used to mediate homologous recombination whereby the marker gene replaces at least a portion of the target dual-role hexokinase gene. In embodiments, the selectable marker can be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the dual-role hexokinase gene without reactivating the latter. In embodiments, the site-specific recombination leaves behind a recombination site which disrupts expression of the dual-role hexokinase protein. In other embodiments, the homologous recombination vector can be constructed to also leave a deletion in the dual-role hexokinase gene following excision of the selectable marker, as is well known to one skilled in the art.

In other embodiments, deletions can be made to a dual-role hexokinase target gene using mitotic recombination as described by Wach at al. (Yeast, 10:1793-1808; 1994). Such a method can involve preparing a DNA fragment that contains a selectable marker between genomic regions that can be as short as 20 bp, and which bound a target DNA sequence. In other embodiments, this DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. In embodiments, the linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence (as disclosed, for example, in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).

Moreover, promoter replacement methods can be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described by Mnaimneh et al. ((2004) Cell 118(1):31-44).

In other embodiments, the dual-role hexokinase target gene encoded activity can be disrupted using random mutagenesis, which can then be followed by screening to identify strains with reduced or substantially eliminated activity. In this type of method, the DNA sequence of the target gene encoding region, or any other region of the genome affecting carbon substrate dependency for growth, need not be known, in embodiments, a screen for cells with reduced dual-role hexokinase activity, or other mutants having reduced dual-role hexokinase activity, can be useful as recombinant host cells of the invention.

Methods for creating genetic mutations are common and well known in the art and can be applied to the exercise of creating mutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.

Chemical mutagenesis of host cells can involve, but is not limited to, treatment with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). Such methods of mutagenesis have been reviewed in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, chemical mutagenesis with EMS can be performed as disclosed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, the introduction of a mutator phenotype can also be used to generate random chromosomal mutations in host cells. In embodiments, common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAG1, RAD18 or RAD1. In other embodiments, restoration of the non-mutator phenotype can be obtained by insertion of the wildtype allele. In other embodiments, collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced or eliminated dual-role hexokinase activity.

Genomes have been completely sequenced and annotated and are publicly available for the following yeast strains: Ashbya gossypii ATCC 10895, Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c, Schizosaccharomyces pombe 972h-, and Yarrowia lipolytica CLIB122. Typically BLAST (described above) searching of publicly available databases with known dual-role hexokinase polynucleotide or polypeptide sequences, such as those provided herein, is used to identify candidate dual-role hexokinase-encoding sequences of other host cells, such as yeast cells.

Accordingly, it is within the scope of the invention to provide dual-role hexokinase polynucleotides, genes and polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of the hexokinase polynucleotides or polypeptides disclosed herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

The modification of a dual-role hexokinase in a recombinant host cell disclosed herein to reduce or eliminate dual-role hexokinase activity can be confirmed using methods known in the art. For example, one can screen for disruption of hexokinase 2 in S. cerevisiae by PCR (for example, looking for lack of a PCR product with primers such as those listed in Example 2) or by Southern blotting using a probe designed to the hexokinase 2 sequence. Alternatively, one can screen for decreased glucose consumption and higher yield of biomass which is phenotypically indicative of a hexokinase 2 disruption.

Introduction of Hexose Kinase Activity

Applicants have found that the inclusion of a heterologous polynucleotide encoding a polypeptide having hexose kinase activity in a recombinant host cell comprising a modification in an endogenous polynucleotide, gene or polypeptide having dual-role hexokinase activity wherein the activity of the dual-role hexokinase is reduced or eliminated can result in altered glucose repression in the recombinant host cell. The introduction of a heterologous polynucleotide encoding a polypeptide having hexose kinase activity may result in an improved redox balance, increased glucose consumption and/or increased product formation by a pyruvate-utilizing biosynthetic pathway.

Hexose kinase polynucleotides, genes or polypeptides known in the art or that are identified as disclosed herein can be expressed in a recombinant host cell disclosed herein.

Suitable hexose kinase polypeptides include, but are not limited to those that are typically dual-role hexokinases in the host cell but have been modified to reduce or eliminate the glucose repression function. Such hexose kinase polypeptides may be encoded by a polynucleotide comprising a conditional promoter such that the expression of the polypeptide is conditional. As an example, dual-role hexokinase polynucleotides, genes and polypeptides in Saccharomyces cerevisiae include, but are not limited to, those in Table 3.

TABLE 3 Example hexose kinases that are dual-role hexokinases in S. cerevisiae Nucleic Protein Nucleic Acid Acid GenBank Protein GenBank SEQ ID Accession SEQ ID Description Accession No. NO No. NO HXK2 Z72775.1 1 CAA96973.1 2 (hexokinase 2) from S. cerevisiae Yarrowia AJ011524.1 114 CAA09674.1 115 lipolytica YIHXK1 Schwanniomyces S78714.1 116 AAB34892.1 117 occidentalis SoXHK Human NM_000162.3 118 NP_000153.1 119 pancreatic glucokinase (hexokinase 4; GCK)

In embodiments, suitable heterologous polynucleotides encode hexose kinases which are dual-function hexokinases in a particular host cell but are expressed in said host cell under the control of a conditional promoter such that glucose repression is altered under conditions where the promoter is not activated or not activated to a significant extent. In embodiments, HXK2 is expressed in S. cerevisiae under the control of a conditional promoter. In embodiments, a dual-function hexokinase having at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 2, 115, 117, or 119 (see Table 3) is encoded in S. cerevisiae by a polynucleotide comprising a conditional promoter sequence. In embodiments, a dual-function hexokinase of SEQ ID NO: 2, 115, 117, or 119 (see Table 3) is encoded in S. cerevisiae by a polynucleotide comprising a conditional promoter sequence. In embodiments, the conditional promoter sequence is derived from the OLE1 promoter region. In embodiments, the promoter sequence is at least 95% identical to SEQ ID NO: 98. In embodiments, the promoter sequence comprises SEQ ID NO: 98. In embodiments, the promoter is SNO1 (SEQ ID NO: 140) or SNZ1 (SEQ ID NO: 141). In embodiments, the promoter sequence is at least about 95% identical to SEQ ID NO: 140 or 141.

In embodiments, a polynucleotide encoding a dual-role hexokinase disclosed herein or known in the art can be modified using methods disclosed herein such that the glucose repression activity is reduced or eliminated by altering the cellular localization od rhw wnxosws polypeptide. For example, a decapeptide at the N-terminus of hexokinase 2 (Lys6-Met15) has been implicated as a domain involved with MIG1 binding, and it is believed that the hexokinase 2-MIG1 complex is imported into the nucleus where both genes can function as transcriptional regulators. Ahuatzi et al. describes a Lys6-Met15 deletion mutant of HXK2 could no longer bind MIG1 and was localized to the cytosol and could not enter the nucleus (Ahuatzi et al. (2004) The Glucose-regulated Nuclear Localization of Hexokinase 2 in Saccharomyces cerevisiae Is Mig1-dependent. JBC 279(14):14440-6).

Thus, deletion or mutation of the MIG1-interaction domain from hexokinase 2 (or related hexokinases) using molecular biology methods known in the art would allow the enzyme to function as a glycolytic enzyme but prevent the enzyme from being translocated to the nucleus and functioning as a transcriptional regulator. In a recombinant host cell comprising reduced or substantially eliminated hexokinase 2 activity, with this modification, one could obtain the growth benefit of the hexokinase 2 reduction, but also high glucose uptake rates akin to the wildtype strain. Therefore, provided herein is a heterologous polynucleotide encoding a polypeptide having hexose kinase activity comprising a mutation or deletion in a protein binding domain necessary for nuclear translocation. In embodiments, the domain is the MIG1-interaction domain. In embodiments, the polynucleotide has at least about 85%, at least about 90%, or at least about 95% identity to SEQ ID NO: 132. In embodiments, the polynucleotide is SEQ ID NO: 132. In embodiments, the polypeptide has at least about 85%, at least about 90%, or at least about 95% identity to SEQ ID NO: 130. In embodiments, the polypeptide is SEQ ID NO: 130.

In embodiments, a heterologous polynucleotide encoding a polypeptide having hexose kinase activity is overexpressed, or expressed at a level that is higher than endogenous expression of the same or related endogenous gene, if any. In other embodiments, a polypeptide having hexose kinase activity is native to a recombinant host cell. In other embodiments, a polypeptide having hexose kinase activity is not native to a recombinant host cell.

In embodiments, the heterologous polynucleotide encoding a polypeptide having hexose kinase activity comprises a constitutive promoter sequence. In embodiments, the constitutive promoter sequence is derived from the ADH1 promoter region. In embodiments, the constitutive promoter sequence has at least 95% identity to SEQ ID NO: 131. In embodiments, the constitutive promoter sequence is SEQ ID NO: 131.

In embodiments, a polypeptide having hexose kinase activity catalyzes the conversion of hexose to hexose-6-phosphate. In other embodiments, a polypeptide having hexose kinase activity catalyzes the conversion of D-glucose to D-glucose 6-phosphate, D-fructose to D-fructose 6-phosphate, and/or D-mannose to D-mannose 6-phosphate.

In embodiments, such a polynucleotide, gene and/or polypeptide can be K. lactis RAG5, H. polymorpha HPGLK1, S. pombe HXK2, or combinations thereof.

In embodiments, a polynucleotide, gene and/or polypeptide encoding hexose kinase activity corresponds to the Enzyme Commission Number EC 2.7.1.1. In other embodiments, a polynucleotide, gene and/or polypeptide encoding hexose kinase can include, but is not limited to, a sequence selected from the following Table 4 or from Table 3. Hexose kinases suitable for expression in S. cerevisiae include those disclosed in Table 4. The hexose kinases disclosed in Table 4 are not dual-function hexokinases when expressed in S. cerevisiae, but one of skill in the art will recognize that certain of the hexose kinases suitable for expression in S. cerevisiae, will be dual-function hexokinases in other types of host cells.

TABLE 4 Example hexose kinase coding regions and proteins and source organism Target Nucleic gene and Nucleic Acid acid Amino acid Protein source GenBank Accession SEQ ID GenBank SEQ ID organism No. or Gene ID No. NO: Accession No. NO: RAG5 from NC_006040 3 XP_453567 4 K. lactis REGION: 973371..974828 HPGLK1 AY034434 5 AAK60444 6 from H. polymorpha HXK2 from X92895 7 NP_593865 8 S. pombe S. cerevisiae Entrez GeneID: 120 — 121 HXK1 850614 S. cerevisiae Entrez GeneID: 122 — 123 GLK1 850317

In other embodiments, a polynucleotide, gene and/or polypeptide encoding a hexose kinase can have at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to that of any one of the sequences of Table 3 or Table 4, wherein such a polynucleotide or gene encodes, or such a polypeptide has, hexose kinase activity. Still other examples of hexose kinase polynucleotides, genes and polypeptides that can be expressed in a recombinant host cell disclosed herein include, but are not limited to, an active variant, fragment or derivative of any one of the sequences of Table 3 or Table 4, wherein such a polynucleotide or gene encodes, or such a polypeptide has, hexose kinase activity. Still other examples of hexose kinase polynucleotides, genes and polypeptides that can be expressed in a recombinant host cell disclosed herein include, but are not limited to, an active variant, fragment or derivative of any one of the sequences of K. lactis KIGLK1 (Nucleic acid SEQ ID NO: 124; Amino acid SEQ ID NO: 125) or Hansenula polymorpha HPHXK1 (Nucleic acid SEQ ID NO: 126; Amino acid SEQ ID NO: 127), wherein such a polynucleotide or gene encodes, or such a polypeptide has, hexose kinase activity.

In other embodiments, a polynucleotide, gene and/or polypeptide encoding hexose kinase can be used to identify another hexose kinase polynucleotide, gene and/or polypeptide sequences and/or can be used to identify a hexose kinase homolog in other cells, as disclosed above for dual-role hexokinases. Such hexose kinase encoding sequences can be identified, for example, in the literature and/or in bioinformatics databases well known to the skilled person. For example, the identification of a hexose kinase encoding sequence in another cell type using bioinformatics can be accomplished through BLAST (as disclosed above) searching of publicly available databases with a known hexose kinase encoding DNA and polypeptide sequence, such as any of those provided herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Modification of Pyruvate Decarboxylase

In embodiments, a recombinant host cell disclosed herein can comprise a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase (PDC) activity or a modification in an endogenous polypeptide having PDC activity. In embodiments, a recombinant host cell disclosed herein can have a modification or disruption of one or more polynucleotides, genes and/or polypeptides encoding PDC. In embodiments, a recombinant host cell comprises a deletion, mutation, and/or substitution in one or more endogenous polynucleotides or genes encoding a polypeptide having PDC activity, or in one or more endogenous polypeptides having PDC activity. Such modifications, disruptions, deletions, mutations, and/or substitutions can result in PDC activity that is reduced or substantially eliminated, resulting, for example, in a PDC knock-out (PDC-KO) phenotype.

In embodiments, the endogenous pyruvate decarboxylase activity of a recombinant host cell disclosed herein converts pyruvate to acetaldehyde, which can then be converted to ethanol or to acetyl-CoA via acetate. In other embodiments, a recombinant host cell is Kluyveromyces lactis containing one gene encoding pyruvate decarboxylase, Candida glabrata containing one gene encoding pyruvate decarboxylase, or Schizosaccharomyces pombe containing one gene encoding pyruvate decarboxylase.

In other embodiments, the recombinant host cell is Saccharomyces cerevisiae containing three isozymes of pyruvate decarboxylase encoded by the PDC1, PDC5, and PDC6 genes, as well as a pyruvate decarboxylase regulatory gene, PDC2. In a non-limiting example in S. cerevisiae, the PDC1 and PDC5 genes, or the PDC1, PDC5, and PDC6 genes, are disrupted. In another non-limiting example in S. cerevisiae, pyruvate decarboxylase activity can be reduced by disrupting the PDC2 regulatory gene. In another non-limiting example, expression of the PDC1 and PDC5 genes, or the PDC1, PDC5, and PDC6 genes are reduced. In another non-limiting example in S. cerevisiae, polynucleotides or genes encoding pyruvate decarboxylase proteins such as those having about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to PDC1, PDC2, PDC5 and/or PDC6 can be disrupted.

In embodiments, a polypeptide having PDC activity or a polynucleotide or gene encoding a polypeptide having PDC activity corresponds to Enzyme Commission Number EC 4.1.1.1. In other embodiments, a PDC gene of a recombinant host cell disclosed herein is not active under the fermentation conditions used, and therefore such a gene would not need to be modified or inactivated.

Examples of a recombinant host cell with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported, such as for Saccharomyces in Flikweert et al. (Yeast (1996) 12:247-257), for Kluyveromyces in Bianchi at al. (Mol. Microbiol. (1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann (Mol. Gen. Genet. (1993) 241:657-666). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC with Accession #200027 and #200028.

Examples of PDC polynucleotides, genes and/or polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, those of the following Table 5.

TABLE 5 Pyruvate decarboxylase target gene coding regions and proteins. SEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid PDC1 pyruvate decarboxylase from 9 10 Saccharomyces cerevisiae PDC5 pyruvate decarboxylase from 11 12 Saccharomyces cerevisiae PDC6 pyruvate decarboxylase from 13 14 Saccharomyces cerevisiae pyruvate decarboxylase from 15 16 Candida glabrata PDC1 pyruvate decarboxylase from 17 18 Pichia stipitis PDC2 pyruvate decarboxylase from 19 20 Pichia stipitis pyruvate decarboxylase from 21 22 Kluyveromyces lactic pyruvate decarboxylase from 23 24 Yarrowia lipolytica pyruvate decarboxylase from 25 26 Schizosaccharomyces pombe pyruvate decarboxylase from 27 28 Zygosaccharomyces rouxii

Other examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, PDC polynucleotides, genes and/or polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of the sequences of Table 5, wherein such a polynucleotide or gene encodes, or such a polypeptide has, Pdc activity. Still other examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, an active variant, fragment or derivative of any one of the sequences of Table 5, wherein such a polynucleotide or gene encodes, or such a polypeptide has, Pdc activity.

In embodiments, a polynucleotide, gene and/or polypeptide encoding a PDC sequence disclosed herein or known in the art can be modified, as disclosed above for hexokinases. In other embodiments, a polynucleotide, gene and/or polypeptide encoding PDC can be used to identify another PDC polynucleotide, gene and/or polypeptide sequence or to identify a PDC homolog in other cells, as disclosed above for hexokinases. Such a PDC encoding sequence can be identified, for example, in the literature and/or in bioinformatics databases well known to the skilled person. For example, the identification of a PDC encoding sequence in other cell types using bioinformatics can be accomplished through BLAST (as described above) searching of publicly available databases with a known PDC encoding DNA and polypeptide sequence, such as those provided herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

The modification of PDC in a recombinant host cell disclosed herein to reduce or eliminate PDC activity can be confirmed using methods known in the art. For example, one can screen for disruption of pyruvate decarboxylase by lack of a PCR product with primers listed in Example 2 or by Southern blotting using a probe designed to a PDC sequence.

Gene Expression in Recombinant Host Cells

Methods for gene expression in recombinant host cells, including, but not limited to, yeast cells are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). In embodiments, the coding region for the hexose kinase genes to be expressed can be codon optimized for the target host cell, as well known to one skilled in the art. Expression of genes in recombinant host cells, including but not limited to yeast cells, can require a promoter operably linked to a coding region of interest, and a transcriptional terminator. A number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, GPM1, and TEF1; and the following inducible promoters suitable for use in yeast: GAL1, GAL10 and CUP1. Suitable for conditional expression is the OLE1 promoter, for which transcription of the gene is induced under anaerobic conditions. While not wishing to be bound by theory, it is believed that anaerobic conditions often prevail during stationary phase, especially in industrial fermentations. Other promoters with stationary-phase expression are known in the art and would also be suitable, such as SNO1 and SNZ1. Suitable transcriptional terminators that can be used in a chimeric gene construct for expression include, but are not limited to, FBA1t, TDH3t, GPM1t, ERG10t, GAL1t, CYC1t, and ADH1t.

Recombinant polynucleotides are typically cloned for expression using the coding sequence as part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a termination control region. The coding region may be from the host cell for transformation and combined with regulatory sequences that are not native to the natural gene encoding hexose kinase. Alternatively, the coding region may be from another host cell.

Vectors useful for the transformation of a variety of host cells are common and disclosed in the literature. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors can comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.

In embodiments, suitable promoters, transcriptional terminators, and hexose kinase coding regions can be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells. Such vectors allow plasmid propagation in both E. coli and yeast strains, and can contain a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast include, but are not limited to, shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2-micron origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are HIS3 (vector pRS423), TRP1 (vector pRS424), LEU2 (vector pRS425) and URA3 (vector pRS426).

In embodiments, construction of expression vectors with a chimeric gene encoding the disclosed hexose kinases can be performed by the gap repair recombination method in yeast. The gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast. In embodiments, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain an approximately 21 bp sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X,” a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g., TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast.

In embodiments, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5″ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding region X-terminator construct is PCR amplified from a plasmid DNA construct and joined to an auxotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.

A recombinant host cell disclosed herein can be cultured using standard laboratory techniques known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). The growth of the recombinant host cells disclosed herein can be measured by methods known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202).

Applicants have provided a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In embodiments, such a recombinant host cell can have an improved redox balance, increased glucose consumption and/or increased formation of a product of a pyruvate-utilizing biosynthetic pathway. As such, Applicants have also provided methods of improving redox balance, increasing glucose consumption and/or increasing formation of a product of a pyruvate-utilizing biosynthetic pathway of a recombinant host cell comprising (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity.

Redox balance and glucose consumption of a recombinant host cell disclosed herein can be measured by methods known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In a non-limiting example, glucose consumption can be measured by quantitating the amount of glucose in culture media by HPLC. Redox balance can be assessed indirectly, for example, by measuring glycerol formation, wherein more glycerol formation implies greater imbalance. Alternatively, redox balance can be assessed by direct analysis of NAD/NADH and NADP/NADPH pools by methods known in the art.

In other embodiments, methods of producing a recombinant host cell are provided comprising (i) providing a recombinant host cell comprising a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (ii) transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (iii) introducing a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity.

In other embodiments, methods for the conversion of hexose into hexose-6-phosphate comprising (i) providing a recombinant host cell comprising a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (ii) transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (iii) introducing a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In other embodiments, methods for the conversion of D-glucose into D-glucose 6-phosphate, D-fructose into D-fructose 6-phosphate, and/or D-mannose into D-mannose 6-phosphate are provided comprising (i) providing a recombinant host cell comprising a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (ii) transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (iii) introducing a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity.

Engineered Biosynthetic Pathways Using Pyruvate.

In embodiments, a recombinant host cell comprising (a) a modification (e.g., a deletion, mutation, and/or substitution) in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification (e.g., a deletion, mutation, and/or substitution) in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity can be engineered to have a biosynthetic pathway for the production of a product of a biosynthetic pathway utilizing pyruvate. Such a recombinant host cell can exhibit an increased production of a product of a biosynthetic pathway utilizing pyruvate. As such, in embodiments, methods for the increased production of a product of a biosynthetic pathway utilizing pyruvate are also provided comprising (i) providing a recombinant host cell comprising (a) a modification (e.g., a deletion, mutation, and/or substitution) in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (ii) growing the recombinant host cell under conditions wherein the product of the pyruvate-utilizing pathway is formed; wherein the amount of product formed by the recombinant host cell is greater than the amount of product formed by a recombinant host cell comprising (a) but not (b).

In other embodiments, methods for the increased production of a product of a biosynthetic pathway utilizing pyruvate are provided comprising (i) providing a recombinant host cell comprising (a) a modification (e.g., a deletion, mutation, and/or substitution) in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and (c) a modification (e.g., a deletion, mutation, and/or substitution) in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity; and (ii) growing the recombinant host cell under conditions wherein the product of the pyruvate-utilizing pathway is formed; wherein the amount of product formed by the recombinant host cell is greater than the amount of product formed by a recombinant host cell comprising (a) and (c) but not (b).

A product from a pyruvate-utilizing biosynthetic pathway used in relation to a recombinant host cell disclosed herein includes, but is not limited to, 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malic acid, isoamyl alcohol, and/or isoprenoids. The features of any pyruvate-utilizing biosynthetic pathway can be engineered in a recombinant host cell disclosed herein in any order. Any product made using a biosynthetic pathway that has pyruvate as the initial substrate can be produced with greater effectiveness in a recombinant host cell disclosed herein. The biosynthetic pathway of a recombinant host cell disclosed herein can be any pathway that utilizes pyruvate and produces a desired product. In some embodiments at least one polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion in biosynthetic pathway is heterologous. In some embodiments, one, two, three, four, or five substrate to product conversions of a biosynthetic pathway are catalyzed by polypeptides encoded by polynucleotides heterologous to the host cell. In some embodiments, the biosynthetic pathway comprises more than one polynucleotide that is heterologous to the yeast cell. In some embodiments, each substrate to product conversion of a biosynthetic pathway is catalyzed by polypeptides encoded by polynucleotides that are heterologous to the host cell. In some embodiments, the polypeptides are heterologous.

An example of a biosynthetic pathway for producing 2,3-butanediol can be engineered in a recombinant host cell disclosed herein, as disclosed in U.S. patent application Ser. No. 12/477,942. The 2,3-butanediol pathway is a portion of the 2-butanol biosynthetic pathway that is disclosed in U.S. Patent Application Publication No. US 2007/0292927 A1 Such pathway steps include, but are not limited to, conversion of pyruvate to acetolactate, for example by acetolactate synthase, conversion of acetolactate to acetoin, for example by acetolactate decarboxylase, and conversion of acetoin to 2,3-butanediol, for example by butanediol dehydrogenase. Butanediol dehydrogenase requires NADH and thereby contributes to redox balance. The skilled person will appreciate that polypeptides having the activity of such pathway steps can be isolated from a variety of sources can be used in the recombinant host cells disclosed herein.

In addition, examples of biosynthetic pathways for production of 2-butanone or 2-butanol that can be engineered in a recombinant host cell disclosed herein are disclosed in U.S. Patent Application Publication Nos. US 2007/0292927 A1 and US 2007/0259410 A1. The pathway in U.S. Patent Application Publication No. US 2007/0292927 A1 is the same as disclosed for butanediol production with the addition of the following steps:

2,3-butanediol to 2-butanone as catalyzed for example by dial dehydratase or glycerol dehydratase; and

2-butanone to 2-butanol as catalyzed for example by butanol dehydrogenase.

Disclosed in U.S. Patent Application Publication No. US 2009/0155870 A1, is the construction of chimeric genes and genetic engineering of yeast for 2-butanol production using the U.S. Patent Application Publication No. US 2007/0292927 A1 disclosed biosynthetic pathway. Further description for gene construction and expression related to these pathways can be found, for example, in International Publication No. WO 2009/046370 (e.g., butanediol dehydratases); and U.S. Patent Application Publication No. US 2009/0269823 A1 (e.g., butanol dehydrogenase) and U.S. Patent Application Publication No. US 20070259410 A1. The skilled person will appreciate that polypeptides having the activity of such pathway steps can be isolated from a variety of sources and can be used in the recombinant host cells disclosed herein.

Examples of biosynthetic pathways for production of isobutanol that can be engineered in a recombinant host cell disclosed herein are also provided in U.S. Patent Application Publication No. US 2007/0092957 A1. As disclosed in U.S. Patent Application Publication No. US 2007/0092957 A1, steps in an example isobutanol biosynthetic pathway include conversion of:

pyruvate to acetolactate as catalyzed by acetolactate synthase

acetolactate to 2,3-dihydroxyisovalerate as catalyzed for example by acetohydroxy acid isomeroreductase, also called ketol-acid reductoisomerase;

2,3-dihydroxyisovalerate to 2-ketoisovalerate as catalyzed for example by acetohydroxy acid dehydratase, also called dihydroxy-acid dehydratase;

2-ketoisovalerate to isobutyraldehyde as catalyzed for example by branched-chain α-keto acid decarboxylase, and

isobutyraldehyde to isobutanol as catalyzed for example by branched-chain alcohol dehydrogenase.

Further description for gene construction and expression related to this pathway can be found, for example, in U.S. Patent Application Publication Nos. US 2008/0261230 A1 and US 2009/0269823 A1. The skilled person will appreciate that polypeptides having the activity of such pathway steps can be isolated from a variety of sources and can be used in a recombinant host cell disclosed herein. Suitable proteins having the ability to catalyze the indicated substrate to product conversions are described in the art. For example, US Published Patent Application Nos. US20080261230 and US20090163376, US20100197519, and U.S. application Ser. No. 12/893,077 describe acetohydroxy acid isomeroreductases; US20070092957 and US20100081154, describe suitable dihydroxyacid dehydratases; suitable alcohol dehydrogenases are described in US Published Patent Application US20090269823 and U.S. Provisional Patent Application No. 61/290,636.

An example of a biosynthetic pathway for production of 1-butanol that can be engineered in a recombinant lost cell disclosed herein is disclosed in U.S. Patent Application Publication No. US 2008/0182308 A1. As disclosed this publication, steps in the disclosed 1-butanol biosynthetic pathway include conversion of:

-   -   acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by         acetyl-CoA acetyltransferase;     -   acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for         example by 3-hydroxybutyryl-CoA dehydrogenase;     -   3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for example         by crotonase;     -   crotonyl-CoA to butyryl-CoA, as catalyzed for example by         butyryl-CoA dehydrogenase;     -   butyryl-CoA to butyraldehyde, as catalyzed for example by         butyraldehyde dehydrogenase; and     -   butyraldehyde to 1-butanol, as catalyzed for example by butanol         dehydrogenase.

Genes that may be used for expression of these enzymes are disclosed, for example, in U.S. Patent Application Publication No. US 2008/0182308 A1, and additional genes that can be used can be identified by one skilled in the art.

An example of a biosynthetic pathway for production of valine that can be engineered in a recombinant host cell disclosed herein includes the steps of acetolactate conversion to 2,3-dihydroxy-isovalerate by acetohydroxyacid reductoisomerase (ILV5), conversion of 2,3-dihydroxy-isovalerate to 2-keto-isovalerate by dihydroxy-acid dehydratase (ILV3), and conversion of 2-keto-isovalerate to valine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). Biosynthesis of leucine includes the same steps to 2-keto-isovalerate, followed by conversion of 2-keto-isovalerate to alpha-isopropylmalate by alpha-isopropylmalate synthase (LEU9, LEU4), conversion of alpha-isopropylmalate to beta-isopropylmalate by isopropylmalate isomerase (LEU1), conversion of beta-isopropylmalate to alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally conversion of alpha-ketoisocaproate to leucine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). It is desired for production of valine or leucine to overexpress at least one of the enzymes in these disclosed pathways.

An example of a biosynthetic pathway for production of isoamyl alcohol that can be engineered in a recombinant host cell disclosed herein includes the steps of leucine conversion to alpha-ketoisocaproate by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1), conversion of alpha-ketoisocaproate to 3-methylbutanal by ketoisocaproate decarboxylase (THI3) or decarboxylase ARO10, and finally conversion of 3-methylbutanal to isoamyl alcohol by an alcohol dehydrogenase such as ADH1 or SFA1, Production of isoamyl alcohol benefits from increased production of leucine or the alpha-ketoisocaproate intermediate by overexpression of one or more enzymes in biosynthetic pathways for these chemicals. In addition, one or both enzymes for the final two steps can be overexpressed.

An example of a biosynthetic pathway for production of lactic acid that can be engineered in a recombinant host cell disclosed herein includes pyruvate conversion to lactic acid by lactate dehydrogenase. Engineering yeast for lactic acid production using lactate dehydrogenase, known as EC 1.1.1.27, is well known in the art such as in Ishida at al. (Appl. Environ. Microbiol. 71:1964-70 (2005)).

An example of a biosynthetic pathway for production of malate that can be engineered in a recombinant host cell disclosed herein includes pyruvate conversion to oxaloacetate by pyruvate carboxylase, and conversion of oxaloacetate to malate by malate dehydrogenase as disclosed in Zelle et al. (Appl. Environ. Microbiol. 74:2766-77 (2008)). In addition, a malate transporter can be expressed.

Examples of biosynthetic pathways for production of isoprenoids can also be engineered in a recombinant host cell disclosed herein. In a non-limiting example, a mevalonate pathway can be used (Martin at al. (2003) Nature Biotech. 21:796-802) which includes the conversion of pyruvate to acetyl-CoA, which is converted to acetoacetyl-CoA, which is converted to 3-hydroxy-3-methylglutaryl-CoA, which is converted to mevalonate and then to isoprenoids. In another non-limiting example, a non-mevalonate pathway is described by Kim and Keisling (Biotechnol. Bioeng, 72:408-15 (2001)).

The skilled person will appreciate that polypeptides having activities of the above-mentioned biosynthetic pathways can be isolated from a variety of sources can be used in a recombinant host cell disclosed herein.

Additional Modifications

Additional modifications that may be useful in cells provided herein include modifications to reduce glycerol-3-phosphate dehydrogenase activity as described in US Patent Application Publication No. 20090305363 (incorporated herein by reference), modifications to a host cell that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in US Patent Application Publication No. 20100120105 (incorporated herein by reference). Yeast strains with increased activity of heterologous proteins that require binding of an Fe—S cluster for their activity are described in US Application Publication No. 20100081179 (incorporated herein by reference). Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway described in U.S. Provisional Application No. 61/380,563 (both referenced provisional applications are incorporated herein by reference in their entirety). Additional modifications that may be suitable for embodiments herein are described in U.S. application Ser. No. 12/893,089.

Additionally, host cells comprising at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis are described in U.S. Provisional Patent Application No. 61/305,333 (incorporated herein by reference), and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphoketolase activity and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphotransacetylase activity are described in US Provisional Patent Application No. 61/356,379.

Growth for Production

A recombinant host cell disclosed herein is grown in fermentation media which contains a suitable carbon substrate. Carbon substrates can include, but are not limited to, monosaccharides such as fructose or galactose, oligosaccharides such as lactose, maltose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates can include ethanol, lactate, succinate, or glycerol.

Additionally a carbon substrate can also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sutter at al., Arch. Microbial. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention can encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, in some embodiments, a carbon substrates can be glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeast cells modified to use C5 sugars. Sucrose can be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose can be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. US 20070031918 A1.

Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipid. Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass can comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media can contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, Yeast Medium (YM) broth, or broth that includes yeast nitrogen base, ammonium sulfate, and dextrose (as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media can also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, can also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between about pH 5.0 to about pH 9.0. In one embodiment, about pH 6.0 to about pH 8.0 can be used for the initial condition. Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 can be used for the initial condition. Suitable pH ranges for the fermentation of other microorganisms are between about pH 3.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 can be used for the initial condition.

Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions can be used for fermentations.

Industrial Batch and Continuous Fermentations

The recombinant host cells disclosed herein can be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas 0. Brock in Biotechnology A Textbook of industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992).

A product of a pyruvate-utilizing biosynthetic pathway related to a recombinant host cell disclosed herein can also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that a product of a pyruvate-utilizing biosynthetic pathway can be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that a recombinant host cell disclosed herein can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.

Methods for Product Isolation from the Fermentation Medium

A product of a pyruvate-utilizing biosynthetic pathway can be isolated from the fermentation medium using methods known in the art for acetone-butanol-ethanol (ABE) fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids can be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the product can be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

Where a product has a low boiling point (e.g., isobutanol), azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation can be used in combination with another separation method to obtain separation around the azeotrope. Methods that can be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so that distillation can be used in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase can be returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase can be further purified by distillation in a second distillation column.

A product of a pyruvate-utilizing biosynthetic pathway can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the product (e.g., isobutanol) can be extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The product-containing organic phase can then be distilled to separate the product from the solvent.

Distillation in combination with adsorption can also be used to isolate a product (e.g., isobutanol) from the fermentation medium. In this method, the fermentation broth containing the product is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREUTP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation can be used to isolate and purify a product (e.g., isobutanol) from the fermentation medium. In this method, the fermentation broth containing the product is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987), and by Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas 0. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. Microbial strains were obtained from The American Type Culture Collection (ATCC), Manassas, Va., unless otherwise noted. The oligonucleotide primers used in the following Examples are given in the following Tables. All the oligonucleotide primers were synthesized by Sigma-Genosys (Woodlands, Tex.) or Integrated DNA Technologies (Coralsville, Iowa).

Synthetic complete medium is described by Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

GC Method

The GC method utilized a ZB-WAXplus column (30 m×0.25 mm ID, 0.25 μm film) from Phenomenex (Torrance, Calif.). The carrier gas was helium at a constant flow rate of 2.3 mL/min; injector split was 1:20 at 250° C.; oven temperature was 70° C. for 1 min, 70° C. to 160° C. at 10° C./min, and 160° C. to 240° C. at 30° C./min. FID detection was used at 260° C. with 40 ml/min helium makeup gas. Culture broth samples were filtered through 0.2 μm spin filters before injection. Depending on analytical sensitivity desired, either 0.1 μl or 0.5 μl injection volumes were used. Calibrated standard curves were generated for the following compounds: ethanol, isobutanol, acetoin, meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. (2S,3S)-2,3-butanediol retention time is 6.8 minutes. meso-2,3-butanediol retention time is 7.2 minutes. Analytical standards were also utilized to identify retention times for isobutyraldehyde, isobutyric acid, and isoamyl alcohol.

HPLC Method

Analysis for glucose and fermentation by-product composition is well known to those skilled in the art. For example, one high performance liquid chromatography (HPLC) method utilizes a Shodex SH-1011 column with a Shodex SH-G guard column (both available from Waters Corporation, Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H₂SO₄ as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol retention time is 47.6 minutes.

The meaning of abbreviations is as follows: “s” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s), “g” means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density, “OD₆₀₀” means the optical density measured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, “% v/v” means volume/volume percent, “wt %” means percent by weight, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography. The term “molar selectivity” is the number of moles of product produced per mole of sugar substrate consumed and is reported as a percent. “SLPM” stands for Standard Liters per Minute (of air), “dO” is dissolved oxygen, Op is “specific productivity” measured in grams isobutanol per gram of cells over time. The term “nt” means nucleotides.

Example 1 Construction of Expression Vectors for Isobutanol Pathway Gene Expression in S. cerevisiae

pLH475-74B8 Construction

The pLH475-Z4B8 plasmid (SEQ ID NO: 29) was constructed for expression of ALS and KARI in yeast. pLH475-Z4B8 is a pHR81 vector (ATCC #87541) containing the following chimeric genes: A) CUP1 promoter region derived sequence (SEQ ID NO: 30), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NOs: 31 and 32) and a CYC1 terminator region derived sequence (“CYC1 terminator 2”; SEQ ID NO: 33); B) ILV5 promoter region derived sequence (SEQ ID NO: 34), Pf5.IlvC-Z4B8 coding region (SEQ ID NOs: 37 and 38) and ILV5 terminator region derived sequence (SEQ ID NO: 35); and C) FBA1 promoter region derived sequence (SEQ ID NO: 36), S. cerevisiae KARI coding region (ILV5; SEQ ID NOs: 39 and 40) and CYC1 terminator region derived sequence.

The Pf5.IlvC-Z4B8 coding region is a sequence encoding KARI derived from Pseudomonas fluorescens with certain mutations, as disclosed in U.S. Patent Application Publication No. US 2009-0163376 A1. More specifically, the Pf5.IlvC-Z4B8 encoded KARI (SEQ ID NO: 38) has the following amino acid changes as compared to the natural Pseudomonas fluorescens KARI:

C33L: cysteine at position 33 changed to leucine, R47Y: arginine at position 47 changed to tyrosine, S50A: serine at position 50 changed to alanine, T52D: threonine at position 52 changed to asparagine, V53A: valine at position 53 changed to alanine, L61F: leucine at position 61 changed to phenylalanine, T80I: threonine at position 80 changed to isoleucine, A156V: alanine at position 156 changed to threonine, and G170A: glycine at position 170 changed to alanine.

The Pf5.IlvC-Z4B8 coding region (SEQ ID NO: 37) was synthesized by DNA 2.0 (Palo Alto, Calif.; based or codons that were optimized for expression in Saccharomyces cerevisiae.

pLH475-JEA1 Construction

The pLH475-JEA1 plasmid (SEQ ID NO:128) was constructed for expression of ALS and KARI in yeast. pLH475-JEA1 is a pHR 81 vector (ATCC #87541) containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO: 30), acetolactate synthase coding region from Bacillus subtilis (AlsS; (SEQ ID NOs: 31 and 32)) and CYC1 terminator 2 (SEQ ID NO: 33)); 2) an ILV5 promoter (SEQ ID NO: 34, Pf5.IlvC-JEA1 coding region and ILV5 terminator (SEQ ID NO: 35); and 3) the FBA1 promoter (SEQ ID NO: 36)S. cerevisiae KARI coding region (ILV5; SEQ ID NOs: 39 and 40) and CYC1 terminator.

The Pf5.IlvC-JEA1 coding region is a sequence encoding KARI derived from Pseudomonas fluorescens with certain mutations, as disclosed in U.S. Patent Application Publication 20090163376A1. More specifically, the Pf5.IlvC-JEA1 encoded KARI (nucleic acid and amino acid sequences of SEQ ID NOs: 41 and 42, respectively) has the following amino acid changes as compared to the natural Pseudomonas fluorescens KARI:

Y24F: tyrosine at position 24 changed to phenylalanine C33L: cysteine at position 33 changed to leucine, R47P: arginine at position 47 changed to praline, S50F: serine at position 50 changed to phenylalanine, T52D: threonine at position 52 changed to asparagine, L61F: leucine at position 61 changed to phenylalanine, T80I: threonine at position 80 changed to isoleucine, A156V: alanine at position 156 changed to threonine. Expression Vector pLH468

The pLH468 plasmid (SEQ ID NO: 43) was constructed for expression of DHAD, KivD and HADH in yeast. Coding regions for Lactococcus lactis ketoisovalerate decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADI-1) were synthesized by DNA2.0 based on codons that were optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 44 and 45) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are (SEQ ID NOs 47 and 46, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426::P_(TDH3)-kivDy-TDH3t), vector pNY8 (SE 0 ID NO: 48; also named pRS426.GPD-ald-GPDt, disclosed in U.S. Patent Application Publication No. US 2008/0182308 A1, Example 17) was digested with AscI and SfiI enzymes, thus excising the GPD promoter region derived sequence and the ald coding region. A TDH3 promoter region derived sequence fragment (SEQ ID NO: 49) from pNY8 was PCR amplified to add an AscI site at the 5′ end, and an SpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067 (SEQ ID NO: 50 and 51). The AscI/SfiI digested pNY8 vector fragment was ligated with the TDH3 promoter PCR product digested with AscI and SpeI, and the SpeI-SfiI fragment containing the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. The triple ligation generated vector pLH467 (pRS426::P_(TDH3)-kivDy-TDH3t). pLH467 (SEQ ID NO: 142) was verified by restriction mapping and sequencing.

pLH435 (pRS425::P_(GPM1)-Hadhy-ADH1t) was derived from vector pRS425::GPM-sadB (SEQ ID NO: 52) which is disclosed in U.S. Provisional Patent Application No. 61/058,970, Example 3. pRS425::GPM-sadB is the pRS425 vector (ATCC #77106) with a chimeric gene containing a GPM1 promoter region derived sequence (SEQ ID NO: 53), a coding region from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB; SEQ ID NO: 55, disclosed in U.S. Patent Application No. 61/048,291; amino acid SEQ ID NO: 56), and an ADH1 terminator region derived sequence (SEQ ID NO: 54). pRS425::GPMp-sadB contains BbvI and PacI sites at the 5′ and 3′ ends of the sadB coding region, respectively. A NheI site was added at the 5′ end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NO: 57 and 58) to generate vector pRS425-GPMp-sadB-NheI, which was verified by sequencing. pRS425::P_(GPMP)-sadB-NheI was digested with NheI and PacI to drop out the sadB coding region, and ligated with the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadny-DNA2.0 to create pLH435 (SEQ ID NO: 143).

To combine KivD and HADH expression cassettes in a single vector, yeast vector pRS411 (ATCC #87474) was digested with SacI and NotI, and ligated with the SacI-SalI fragment from pLH467 that contains the P_(TDH3)-kivDy-TDH3t cassette together with the SalI-NotI fragment from pLH435 that contains the P_(GPM1)-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the vector pRS411::P_(TDH3)-kivDy-P_(GPM1)-Hadhy (pLH441 SEQ ID NO: 144), which was verified by restriction mapping.

In order to generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy and Hadhy, we used pRS423 FBA ilvD(Strep) (SEQ ID NO: 59), which is disclosed in U.S. Patent Application No. 61/100,792, as the source of the ilvD gene. This shuttle vector contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector has an FBA1 promoter region derived sequence (nt 2111 to 3108; ((SEQ ID NO: 36) and FBA1 terminator region derived sequence (nt 4861 to 5860; SEQ ID NO: 60). In addition, it carries the HIS3 marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; (ilvD coding region of vector is SEQ ID NO: 62 and wild-type protein sequence of ilvD is SEQ ID NO: 63) from Streptococcus mutans UA159 (ATCC #700610) is between the FBA1 promoter region derived sequence and FBA1 terminator region derived sequence forming a chimeric gene for expression. In addition there is a lumio tag fused to the ilvD coding region (nt 4829-4849).

The first step was to linearize pRS423::FBA ilvD(Strep) (also called pRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio) with SacI and SacII (with SacII site blunt ended using T4 DNA polymerase), to give a vector with total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI site blunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment. This fragment was ligated with the 9,482 bp vector fragment from pRS423-FBA(SpeI)-ilvD(Streptococcus mutans)-Lumio. This generated vector pLH468 (pRS423::P_(FBA1)-ilvD(Strep)Lumio-FBA1t-P_(TDH3)-kivDy-TDH3t-P_(GPM1)-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.

Example 2 Pyruvate Decarboxylase and Hexokinase 2 Gene Inactivation

This example describes insertion-inactivation of endogenous PDC1, PDC5, and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase. The resulting PDC inactivation strain was used as a host for expression vectors pLH475-Z4B8 and pLH468 that were described in Example 1.

Construction of pdc6::P_(GPM1)-sadB Integration Cassette and PDC6 Deletion:

A pdc6::P_(GPM1)-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO: 64) from pRS425::GPM-sadB (described above) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO: 65) contains the URA3 marker from pRS426 (ATCC #77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 114117-11A through 114117-11D (SEQ ID NOs: 66-69), and 114117-13A and 114117-13B (SEQ ID NOs: 70 and 71). The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′-50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC #200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 72 and 73), and 112590-34F and 112590-49E (SEQ ID NOs: 74 and 75) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD URA-media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::P_(GPM1)-sadB-ADH1t.

Construction of Pdc1:: P_(PDC1)-ilvD Integration Cassette and PDC1 Deletion:

A pdc1:: P_(PDC1)-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO: 76) from pLH468 (described above) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 114117-27A through 114117-27D (SEQ ID NOs: 77-80).

The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′-50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::P_(GPM1)-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs:82 and 83), and primers 112590-49E and 112590-30F (SR1ID NOs: 75 and 129) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6::P_(GPM1)-sadB-ADH1t pdc1::P_(PDC1)-ilvD-FBA1t.

HIS3 Deletion

To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 81). URA3r2 contains the URA3 marker from pRS426 (ATCC #77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion DNA polymerase and primers 114117-45A and 114117-45B (SEQ ID NOs: 84 and 85) which generated a ˜2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain, called NYLA73, has the genotype: BY4700 pdc6:: P_(GPM1)-sadB-ADH1t pdc1:: P_(PDC1)-ilvD-FBA1t Δhis3.

Construction of pdc5::kanMX Intergration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs: 86 and 87) which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 88 and 89). The identified correct transformants have the genotype: BY4700 pdc6::P_(GPM1)-ilvD-sadB-ADH1t pdc1::P_(PDC1)-ilvD-FBA1t Δhis3 pdc5::kanMX4. The strain was named NYLA74.

Deletion of Hexokinase 2:

A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (described above) using Phusion DNA polymerase and primers 384 and 385 (SEQ ID NOs: 90 and 91) which generated a ˜2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5′ region upstream of the HXK2 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HXK2 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region using primers N869 and N871 (SEQ ID NO: 92 and 93). The URA3r2 marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth, and by PCR to verify correct marker removal using primers N946 and N947 (SEQ ID NO: 94 and 95). The resulting identified strain named NYLA83 has the genotype: BY4700 pdc6:: P_(GPM1)-sadB-ADH1t pdc1::P_(PDC1)-ilvD-FBA1t Δhis3 Δhxk2.

Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion

A pdc5::kanMX4 cassette was PCR-amplified as described above. The PCR fragment was transformed into NYLA83, and transformants were selected and screened as described above. The identified correct transformants named NYLA84 have the genotype: BY4700 pdc6:: P_(GPM1)-sadB-ADH1t pdc1::P_(PDC1)-ilvD-FBA1 t Δhis3 Δhxk2 pdc5::kanMX4.

Plasmid vectors pLH468 and pLH475-Z4B8 were simultaneously transformed into strain NYLA84 (BY4700 pdc6::P_(GPM1)-sadB-ADH1t pdc1::P_(PDC1)-ilvD-FBA1t Δhis3 zihxk2 pdc5::kanMX4) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the resulting strain was maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol at 30° C.

Example 3 Production of Isobutanol

The purpose of this example is to describe the production of isobutanol in the yeast strain NYLA84. The yeast strain comprises deletions of PDC1, PDC5, and PDC6, genes encoding three isozymes of pyruvate decarboxylase, and constructs for heterologous expression of AlsS (acetolactate synthase), KARI (keto acid reductoisomerase), DHAD (dihydroxy acid dehydratase), KivD (ketoisovalerate decarboxylase), and SadB (secondary alcohol dehydrogenase).

Strain Construction

Plasmids pLH468 and pLH475-Z4B8 were introduced into NYLA74 or NYLA84, described in Example 2, by standard PEG/lithium acetate-mediated transformation methods. Transformants were selected on synthetic complete medium lacking glucose, histidine and uracil. Ethanol (1% v/v) was used as the carbon source. After three days, transformants were patched to synthetic complete medium lacking histidine and uracil supplemented with both 2% glucose and 1% ethanol as carbon sources. Fermentation seed vials were made by inoculation of cultures into synthetic complete medium lacking histidine and uracil supplemented with both 0.2% glucose and 0.5% ethanol. Glycerol was added to final concentration of 15% (v/v) and vials were stored at −80° C.

Production of Isobutanol

Fermentation inoculum was grown in synthetic complete medium lacking histidine and uracil supplemented with 1% ethanol as a carbon source at 30° C. and shaking at 250 rpm. Inoculation volume for the fermenters was 80 ml. The 80 ml of inoculum in the 800 ml fermentation medium described below resulted in the presence of 0.1% ethanol.

The NYLA84/pLH468+pLH475-Z4B8 strain fermenter was prepared and sterilized with 0.4 L water. After cooling, filter sterilized media was added to give the following final concentrations in 800 mL post-inoculation:

Medium (Final Concentration):

-   6.7 g/L Yeast Nitrogen Base w/o amino acids (Difco) -   2.8 g/L Yeast Synthetic Drop-out Medium Supplement Without     Histidine, Leucine, Tryptophan and Uracil (Sigma Y2001) -   20 mL/L of 1% (w/v) L-Leucine -   4 mL/L of 1% (w/v) L-Tryptophan -   10 g/L glucose -   1 mL/L 1% ergosterol in 50% (v/v) Tween-80/ethanol solution -   0.2 mL/L Sigma DF204 antifoam

The fermenter was set to control at pH 5.5 with KOH, initial dO (dissolved oxygen) 30% by stirring, temperature 30° C., and airflow of 0.2 SLPM (or, 0.25 vvm). At inoculation, the airflow was set to 0.01 SLPM initially, then increased to 0.2 SLPM. Glucose was maintained at 5-15 g/L throughout.

The NYLA74/pLH468 pLH475-Z4B8 strain fermenter was prepared as for the NYLA84/pLH468 pLH475-Z4B8 strain fermenter except that 1 mL/L ergosterol/tween/ethanol solution and 0.2 mL/L Sigma DF204 antifoam were omitted, and glucose was 2 g/L. Initial ethanol concentration in the fermenter was 0.1%.

The fermenter was set to control at pH 5.5 with KOH, initial dO 30% by stirring, temperature 30° C., and airflow of 0.2 SLPM (or, 0.25 vvm). At inoculation, the airflow was set to 0.01 SLPM initially, then increased to 0.2 SLPM. Glucose was maintained at 0.1-2 g/L throughout.

Samples were taken periodically and measured for growth by OD600, and for isobutanol content by HPLC as described in General Methods. FIG. 1 shows the results comparing strains with and without hexokinase 2 deletion for growth (1A) and isobutanol production (1B).

FIG. 2 shows a comparison of growth and isobutanol production for the strain without hexokinase 2 deletion (2A) and the strain with hexokinase 2 deletion (2B). FIG. 3 plots the results as “specific productivity” (Qp) measured in grams isobutanol per gram of cells over time. For the strain without deletion of hexokinase 2, the cell specific productivity dropped from 60-90 hours when there was no longer growth, while for hexokinase 2 deletion strain, the specific productivity was relatively well maintained from 60-140 hours showing that the strain is capable of better non-growth associated production.

Example 4 Prophetic Regulated Expression of Hexokinase in a S. cerevisiae Strain Devoid of Pyruvate Decarboxylase and Hexokinase 2 Activity

This example describes insertion of hexokinase enzyme under a controlled expression in a S. cerevisiae strain where pyruvate decarboxylase (Δpdc1/5/6) and hexokinase 2 (Δhxk2) activity have been removed. Creation of the NYLA84 (Δpdc1/5/6 Δhxk2) strain was described in Example 2.

The HXK2 gene and native terminator from S. cerevisiae (SEQ ID NO: 101) was PCR amplified from genomic DNA from strain BY4700 (ATCC #200866) using Phusion DNA polymerase and primers LA588 (SEQ ID NO: 96) and LA589 (SEQ ID NO: 97), and digested with XbaI and BamHI restriction enzymes. The OLE1 promoter region derived sequence (SEQ ID NO: 98) was PCR amplified from BY4700 genomic DNA using Phusion DNA polymerase and primers LA586 (SEQ ID NO: 99) and LA587 (SEQ ID NO: 100), and digested with HindIII and XbaI restriction enzymes. The HXK2 and P_(OLE1) products were ligated and subcloned into pUC19::loxP-URA3-loxP which was previously digested with HindIII and BamHI. pUC19::loxP-URA3-loxP (SEQ ID NO: 102) contains the URA3 marker from (ATCC #77107) flanked by loxP recombinase sites. The resulting vector was named pLA25 (SEQ ID NO: 103).

The RAG5 gene from K. lactis (SEQ ID NO: 3) was PCR amplified from genomic DNA from strain GG799 (#C1001S, New England Biolabs, Ipswich, Mass.) using Phusion DNA polymerase and primers LA593 and LA594 (SEQ ID NO: 104 and 105), and was digested with HindIII and XbaI restriction enzymes. The gel-purified RAG5 product was ligated with the OLE1 promoter region derived sequence from above, and subcloned into pUC19::loxP-URA3-loxP which was previously digested with HindIII and BamHI. The resulting vector was named pLA31 (SEQ ID NO: 106).

In order to integrate into the TRP1 locus, the P_(OLE1)-HXK2-loxP-URA3-loxP and P_(OLE1)-RAG5-loxP-URA3-loxP cassettes is PCR amplified from plasmids pLA25 and pLA31 using Phusion DNA polymerase and primers BK600 and BK601 (SEQ ID NOs: 107 and 108). The TRP1 portion of each primer is derived from the 5′ region upstream of the TRP1 promoter and 3° region downstream of the coding region such that integration of the P_(OLE1)-HXK2-loxP-URA3-loxP or P_(OLE1)-RAG5-loxP-URA3-loxP cassette results in replacement of the TRP1 coding region. The PCR product is transformed into NYLA84 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants are selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants are screened by PCR to verify correct integration at the TRP1 locus with replacement of the TRP1 coding region using primers 112590-49E (SEQ ID NO: 75) and LA606 (SEQ ID NO: 109). The URA3 marker is recycled by transformation with pRS423::P_(GAL1)-cre (SEQ ID NO: 110) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. Colonies are patched onto YP (1% galactose) plates at 30° C. to induce URA3 marker excision and are transferred onto YP (1% ethanol) plates at 30° C. for recovery. Removal of the URA3 marker is confirmed by patching colonies from the YP (1% ethanol) plates onto synthetic complete media lacking uracil supplemented with 1% ethanol to verify the absence of growth.

Example 5 Prophetic Constitutive Expression of Hexokinase in a S. cerevisiae Strain Devoid of Pyruvate Decarboxylase and Hexokinase 2 Activity

This example describes insertion of hexokinase enzyme under control of the constitutive ADH1-derived promoter sequence in a S. cerevisiae strain where pyruvate decarboxylase (Δpdc1/5/6) and hexokinase 2 (Δhxk2) activity have been removed. Creation of the NYLA84 (Δpdc1/5/6 Δhxk2) strain was described in Example 2.

The RAG5 gene from K. lactis (SEQ ID NO: 3) was PCR amplified from genomic DNA from strain GG799 (#C1001S; New England Biolabs, Ipswich, Mass.) using Phusion DNA polymerase and primers LA593 and LA594 (SEQ ID NOs: 104 and 105), and was digested with HindIII and XbaI restriction enzymes. The ADH1 promoter region derived sequence (SEQ ID NO: 131) was PCR amplified from BY4700 genomic DNA using Phusion DNA polymerase and primers LA595 and LA597 (SEQ ID NOs: 112 and 113), and digested with HindIII and XbaI restriction enzymes. The gel-purified RAG5 product was ligated with the ADH1 promoter fragment, and subcloned into pUC19::loxP-URA3-loxP which was previously digested with HindIII and BamHI. The resulting vector was named pLA32 (SEQ ID NO: 111).

In order to integrate into the TRP1 locus, the P_(ADH1)-RAG5-loxP-URA3-loxP cassette is PCR amplified from plasmid pLA32 using Phusion DNA polymerase and primers BK600 and BK601 (SEQ ID NOs 107 and 108). The TRP1 portion of each primer is derived from the 5′ region upstream of the TRP1 promoter and 3′ region downstream of the coding region such that integration of the P_(ADH1)-RAG5-loxP-URA3-loxP cassette results in replacement of the TRP1 coding region. The PCR product is transformed into NYLA84 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants are selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants are screened by PCR to verify correct integration at the TRP1 locus with replacement of the TRP1 coding region using primers 112590-49E (SEQ ID NO: 75) and LA606 (SEQ ID NO: 109). The URA3 marker is recycled by transformation with pRS423::P_(GAL1)-cre (SEQ ID NO: 110) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. Colonies are patched onto YP (1% galactose) plates at 30° C. to induce URA3 marker excision and are transferred onto YP (1% ethanol) plates at 30° C. for recovery. Removal of the URA3 marker is confirmed by patching colonies from the YP (1% ethanol) plates onto synthetic complete media lacking uracil supplemented with 1% ethanol to verify the absence of growth.

Example 6 Prophetic Isobutanol Production Using NYLA84 Strains with Regulated Expression of Hexose Kinase Isobutanol Strain and Production

The expression constructs pLH475-JEA1 and pLH468 (described in Example 1) are transformed into strains NYLA84, NYLA84 trp1::POLE1-HXK2 and NYLA84 trp1::POLE1-RAG5 (described in Example 4) by standard PEG/lithium acetate-mediated transformation methods. Transformants are selected on synthetic complete medium lacking glucose, histidine and uracil. Ethanol (1% v/v) is used as the carbon source. After three days, transformants are patched to synthetic complete medium lacking histidine and uracil supplemented with both 2% glucose and 1% ethanol as carbon sources. Seed vials are made by inoculation of cultures into synthetic complete medium lacking histidine and uracil supplemented with both 0.2% glucose and 0.5% ethanol. Glycerol is added to final concentration of 15% (v/v) and vials are stored at −80° C.

Isobutanol Production

Seed vials of NYLA84 pLH475-JEA1, NYLA84 trp1::P_(OLE1)-HXK2, and NYLA84 trp1::P_(OLE1)-RAG5 are inoculated into 80 mL of synthetic complete medium lacking histidine and uracil supplemented with both 0.25% glucose and 0.5% ethanol as carbon sources at 30° C. A 1 liter fermenter is prepared and sterilized with 0.4 L water. After cooling, filter sterilized medium is added to give the following final concentrations in 800 mL post-inoculation:

Medium (Final Concentration):

6.7 g/L, Yeast Nitrogen Base w/o amino acids (Difco) 2.8 g/L, Yeast Synthetic Drop-out Medium Supplement Without Histidine, Leucine, Tryptophan and Uracil (Sigma Y2001) 20 mL/L of 1% (w/v) L-Leucine 4 mL/L of 1% (w/v) L-Tryptophan 1 mL/L ergosterol/tween/ethanol solution 0.2 mL/L Sigma DF204 10 g/L glucose

The fermenter is set to control at pH 5.5 with KOH, 30% dO, temperature 30° C., and airflow of 0.2 SLPM (or, 0.25 vvm). At inoculation, the airflow is set to 0.01 SLPM initially, then increased to 0.2 SLPM once growth was established. Glucose is maintained at 5-15 g/L throughout by manual addition. Alternatively, the fermenter is set to control at pH 5.5 with KOH, 3-5% dO, temperature 30° C., and airflow of 0.2 SLPM (or 0.25 vvm). At inoculation, the airflow is set to 0.01 SLPM initially, increased to 0.2 SLPM once growth is established.

To quantify the loss of isobutanol due to stripping, the off-gas from the fermentor is directly sent to a mass spectrometer (Prima dB mass speatrometer, Thermo Electron Corp., Madison, Wis.) to quantify the amount of isobutanol in the gas stream. The isobutanol peaks at mass to charge ratios of 74 or 42 are monitored continuously to quantify the amount of isobutanol in the gas stream. Glucose and organic acids in the aqueous phase are monitored during the fermentation using HPLC. Glucose is also monitored quickly using a glucose analyzer (YSI, Inc., Yellow Springs, Ohio). Isobutanol and isobutyric acid in the aqueous phase are quantified by HPLC as described in the General Methods Section herein above after the aqueous phase is removed periodically from the fermentor.

Example 7 Prophetic Modification of Hexose Kinase Function

The purpose of this example is to describe how the function of hexose kinase can be altered by deletion of a protein interaction domain that prevents function as a transcriptional regulator. The MIG1-interaction domain (Lys6-Met15) is removed from S. cerevisiae HXK2 which allows function as a glycolytic enzyme but prevents translocation to the nucleus.

In order to remove the N-terminal MIG1-interaction domain from S. cerevisiae HXK2, an integration cassette is constructed using the pUC19::loxP-URA3-loxP plasmid. The gene encoding HXK2 with an internal deletion of the Lys6-Met15 region (bp 19-48) and ADH1 terminator region derived sequence is synthesized by DNA 2.0 with codon-optimization for S. cerevisiae (SEQ ID NO: 132). The HXK2(ΔLys6-Met15)-ADH1t cassette is PCR-amplified using Phusion DNA polymerase and primers E001 and E002 (SEQ ID NOS: 133 and 134) and subcloned into pUC19::loxP-URA3-loxP via HindIII BamHI sites, creating plasmid pUC19::loxP-URA3-loxP-HXK2(Lys6-Met15)-ADH1t (SEQ ID NO: 139).

The HXK2(ΔLys6-Met15)ADH1t-loxP-URA3-loxP cassette is PCR amplified using Phusion DNA polymerase and primers E003 and E004 (SEQ ID NOS: 135 and 136). Primer E003 contains sequence from the HXK2 promoter region and primer E004 contains sequence from the HXK2 terminator, such that integration of the HXK2(ΔLys6-Met15)ADH1t-loxP-URA3-loxP cassette results in replacement of the native HXK2 coding sequence. The PCR product is transformed into NYLA74 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants are selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants are screened by PCR to verify correct integration at the HXK2 locus using primers E005 and E006 (SEQ ID NOS: 137 and 138). The URA3 marker is recycled by transformation with pRS423::P_(GAL1)-cre (SEQ ID NO: 110) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. Colonies are patched onto YP (1% galactose) plates at 30° C. to induce URA3 marker excision and are transferred onto YP (1% ethanol) plates at 30° C. for recovery. Removal of the URA3 marker is confirmed by patching colonies from the YP (1% ethanol) plates onto synthetic complete media lacking uracil supplemented with 1% ethanol to verify the absence of growth.

Example 8 Prophetic Isobutanol Production Using NYLA44 Strains with Modified Function of Hexose Kinase Isobutanol Strain and Production

The expression constructs pLF1475-JEA1 and pLH468 (described in Example 1) are transformed into strains NYLA74 hxk2Δ::HXK2(ΔLys6-Met15) (described in Example 7) by standard PEG/lithium acetate-mediated transformation methods. Transformants are selected on synthetic complete medium lacking glucose, histidine and uracil. Ethanol (1% v/v) is used as the carbon source. After three days, transformants are patched to synthetic complete medium lacking histidine and uracil supplemented with both 2% glucose and 1% ethanol as carbon sources. Seed vials are made by inoculation of cultures into synthetic complete medium lacking histidine and uracil supplemented with both 0.2% glucose and 0.5% ethanol. Glycerol is added to final concentration of 15% (v/v) and vials are stored at −80° C.

Isobutanol Production

Seed vials of NYLA74 hxk2Δ::HXK2(ΔLys6-Met15) pLH468 pLH475-JEA1 are inoculated into 80 mL of synthetic complete medium lacking histidine and uracil supplemented with both 0.25% glucose and 0.5% ethanol as carbon sources at 30° C. A 1 liter fermenter is prepared and sterilized with 0.4 L water. After cooling, filter sterilized medium is added to give the following final concentrations in 800 mL post-inoculation:

Medium (Final Concentration):

6.7 g/L, Yeast Nitrogen Base w/o amino acids (Difco) 2.8 g/L, Yeast Synthetic Drop-out Medium Supplement Without Histidine, Leucine, Tryptophan and Uracil (Sigma Y2001) 20 mL/L of 1% (w/v) L-Leucine 4 mL/L of 1% (My) L-Tryptophan 1 mL/L ergosterol/tween/ethanol solution 0.2 mL/L Sigma DF204 10 g/L glucose

The fermenter is set to control at pH 5.5 with KOH, 30% dO, temperature 30° C., and airflow of 0.2 SLFM (or, 0.25 vvm). At inoculation, the airflow is set to 0.01 SLPM initially, then increased to 0.2 SLPM once growth was established. Glucose is maintained at 5-15 g/L throughout by manual addition. Alternatively, the fermenter is set to control at pH 5.5 with KOH, 3-5% dO, temperature 30° C., and airflow of 0.2 SLPM (or 0.25 vvm). At inoculation, the airflow is set to 0.01 SLPM initially, increased to 0.2 SLPM once growth is established.

To quantify the loss of isobutanol due to stripping, the off-gas from the fermentor is directly sent to a mass spectrometer (Prima dB mass spectrometer, Thermo Electron Corp., Madison, Wis.) to quantify the amount of isobutanol in the gas stream. The isobutanol peaks at mass to charge ratios of 74 or 42 are monitored continuously to quantify the amount of isobutanol in the gas stream. Glucose and organic acids in the aqueous phase are monitored during the fermentation using HPLC. Glucose is also monitored quickly using a glucose analyzer (YSI, Inc., Yellow Springs, Ohio). Isobutanol and isobutyric acid in the aqueous phase are quantified by HPLC as described in the General Methods Section herein above after the aqueous phase is removed periodically from the fermenter. 

1. A recombinant yeast host cell comprising: (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in the host cell wherein the activity of the polypeptide of (a) is reduced or substantially eliminated; and (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity.
 2. The recombinant host cell of claim 1 wherein the glucose consumption rate is increased as compared to that of the host cell comprising (a) but not (b).
 3. The recombinant host cell of claim 1 wherein the modification of (a) is a deletion.
 4. The recombinant host cell of claim 1 wherein the recombinant host cell further comprises (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity.
 5. The recombinant host cell of claim 4 wherein pyruvate decarboxylase activity is reduced or substantially eliminated.
 6. The recombinant host of claim 1 wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is S. cerevisiae.
 7. The recombinant host cell of claim 1, wherein the polypeptide of (a) is RAG5, and wherein the recombinant host cell is K. lactis, or wherein the polypeptide of (a) is HPGLK1, and wherein the recombinant host cell is H. polymorpha, or wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is S. pombe. 8-9. (canceled)
 10. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) comprises the polypeptide of (a) having a deletion of a protein interaction domain that prevents function as a transcriptional regulator.
 11. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 2, 115, 117, 119, 4, 6, 8, 121, or
 123. 12. The recombinant host cell of claim 6 wherein the heterologous polynucleotide of (b) encodes a polypeptide of SEQ ID NO: 4, 6, 8, 121, or
 123. 13. The recombinant host cell of claim 5 wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO: 4, 6, 8, 121, or
 123. 14. The recombinant host cell of claim 5 wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO:
 130. 15. The recombinant host cell of claim 1 wherein the polypeptide encoded by the heterologous polynucleotide of (b) is constitutively expressed.
 16. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) comprises i) a promoter region derived from the S. cerevisiae ADH1 promoter region or ii) a promoter region having at least about 85% identity to SEQ ID NO:
 131. 17. The recombinant host cell of claim 6 wherein the heterologous polynucleotide of (b) comprises a conditional promoter and encodes a polypeptide having at least 85% identity to SEQ ID NO: 4 or SEQ ID NO:
 2. 18. The recombinant host cell of claim 17 wherein the conditional promoter comprises a sequence derived from the OLE1 promoter region.
 19. The recombinant host cell of claim 1 further comprising a pyruvate utilizing biosynthetic pathway which forms a product selected from the group consisting of: 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malate, isoamyl alcohol, and isoprenoids.
 20. The recombinant host cell of claim 19 wherein the product is isobutanol. 21-26. (canceled)
 27. A method of increasing glucose consumption of a recombinant host cell comprising: (i) providing the recombinant host cell of claim 1; and (ii) growing the recombinant host cell under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form; wherein the glucose consumption of the recombinant host cell is greater than the glucose consumption of a host cell comprising (a) but not (b).
 28. A method of increasing the formation of a product of a pyruvate-utilizing biosynthetic pathway comprising; (i) providing the recombinant host cell of claim 19, or combinations thereof; and (ii) growing the recombinant host cell under conditions wherein the product of the pyruvate-utilizing pathway is formed; wherein the amount of product formed by the recombinant host cell is greater than the amount of product formed by a host cell comprising (a) but not (b).
 29. The method of claim 28 wherein the product is butanol.
 30. The butanol of claim 29, wherein said butanol is isobutanol.
 31. (canceled) 